Humanized rodents that express heavy chains containing VL domains

Abstract

Non-human animals, tissues, cells, and genetic material are provided that comprise a modification of an endogenous non-human heavy chain immunoglobulin sequence and that comprise an ADAM6 activity functional in a rodent (e.g., a mouse), wherein the non-human animals rearrange human immunoglobulin light chain gene segments in the context of heavy chain constant regions and express immunoglobulin-like molecules comprising human immunoglobulin light chain variable domains fused to heavy chain constant domains that are cognate with human immunoglobulin light chain variable domains fused to light chain constant do- mains.

Description

HUMANIZED RODENTS THAT EXPRESS HEAVY CHAINS CONTAINING VL DOMAINS FIELD OF INVENTION

[0001] Genetically modified non-human fertile animals that express human immunoglobulin-like binding proteins comprising an immunoglobulin heavy chain constant region fused with an immunoglobulin light chain variable domain are provided, as well as binding proteins having an immunoglobulin light chain variable domain fused to a light chain constant domain and an immunoglobulin light chain variable domain fused to a heavy chain constant domain. Genetically modified mice, cells, embryos, and tissues that comprise a nucleic acid sequence that encodes a functional ADAM6 protein are described, wherein the mice, cells, embryos, and tissues comprise human immunoglobulin light chain gene segments operably linked to one or more non-human immunoglobulin heavy chain constant genes. Modifications include human and / or humanized immunoglobulin loci. Mice that comprise ADAM6 function are described, including mice that comprise an ectopic nucleic acid sequence that encodes an ADAM6 protein. Genetically modified male mice that comprise a genetic modification of an endogenous mouse immunoglobulin V<com:Sub>H< / com:Sub> region locus, and that further comprise ADAM6 activity are described, including mice that comprise an ectopic nucleic acid sequence that restores or maintains fertility to the male mouse. Exemplary fertility is fertility that is comparable to wild-type mice.

[0002] Genetically modified non-human fertile animals that comprise a deletion or a modification of an endogenous ADAM6 gene or homolog or ortholog thereof, and that comprise a genetic modification that restores ADAM6 (or homolog or ortholog thereof) function in whole or in part are provided, wherein the non-human animals express a human immunoglobulin light chain variable sequence in the context of a heavy chain constant sequence. Cells expressing such binding proteins, rodents (e.g., mice) that make them, and related methods and compositions are also provided.

[0003] Genetically engineered animals that express antibodies comprising light chain variable regions fused with heavy chain constant regions, wherein the non-human animals lack a functional endogenous ADAM6 gene but retain ADAM6 function, are described, including rodents (e.g., mice) that comprise a modification of an endogenous immunoglobulin heavy chain variable (V<com:Sub>H< / com:Sub>) region locus that renders the mouse incapable of making a functional ADAM6 protein and results in a loss in fertility. The genetically modified mice comprise an immunoglobulin V<com:Sub>H< / com:Sub> locus characterized by a plurality of human V<com:Sub>L< / com:Sub>, J<com:Sub>L< / com:Sub> and optionally D<com:Sub>H< / com:Sub> gene segments or a combination thereof, and that further comprise ADAM6 function are described, including mice that comprise an ectopic nucleic acid sequence that restores fertility to a male mouse. The genetically modified mice express antibodies that lack heavy chain variable domains and instead comprise variable domains comprising rearranged light chain gene segments.

[0004] Genetically modified rodents (e.g., mice), cells, embryos, and tissues that comprise a nucleic acid sequence encoding a functional ADAM6 locus are described, wherein the mice, cells, embryos, and tissues express an immunoglobulin heavy chain comprising a human light chain variable domain. Further, the mice, cells, embryos, and tissues lack a functional endogenous ADAM6 gene but retain ADAM6 function characterized by the presence of an ectopic nucleic acid sequence that encodes an ADAM6 protein. Methods for making antibody sequences in fertile non-human animals that are useful for binding antigens are provided. BACKGROUND

[0005] Over the last two decades, pharmaceutical applications for antibodies fueled a great deal of research into making antibodies that are suitable for use as human therapeutics. Early antibody therapeutics, based on mouse antibodies, were not ideal as human therapeutics because repeatedly administering mouse antibodies to humans results in immunogenicity problems that can confound long-term treatment regimens. Solutions based on humanizing mouse antibodies to make them appear more human and less mouse- like were developed. Methods for expressing human immunoglobulin sequences for use in antibodies followed, mostly based on in vitro expression of human immunoglobulin libraries in phage, bacteria, or yeast. Finally, attempts were made to make useful human antibodies from human lymphocytes in vitro, in mice engrafted with human hematopoietic cells, and in transchromosomal or transgenic mice with disabled endogenous immunoglobulin loci.

[0006] For the creation of these mice, it was necessary to disable the endogenous mouse immunoglobulin genes so that the randomly integrated fully human transgenes would function as the expressed repertoire of immunoglobulins in the mouse. Such mice can make human antibodies suitable for use as human therapeutics, but these mice display substantial problems with their immune systems. These problems lead to several experimental hurdles, for example, the mice are impractical for generating sufficiently diverse antibody repertoires, require the use of extensive re-engineering fixes, provide a suboptimal clonal selection process likely due to incompatibility between human and mouse elements, and an unreliable source of large and diverse populations of human variable sequences needed to be truly useful for making human therapeutics.

[0007] Transgenic mice that contain fully human antibody transgenes contain randomly inserted transgenes that contain unrearranged human immunoglobulin heavy chain variable sequences (V, D, and J sequences) linked to human heavy chain constant sequences, and unrearranged human immunoglobulin light chain variable sequences (V and J) linked to human light chain constant sequences. The mice therefore generate rearranged antibody genes from loci other than endogenous loci, where the rearranged antibody genes are fully human. In general, the mice contain human heavy chain sequences and human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain sequences, although mice with at least some human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> sequences have also been reported. The transgenic mice generally have damaged and nonfunctional endogenous immunoglobulin loci, or knockouts of endogenous immunoglobulin loci, so that the mice are incapable of rearranging human antibody sequences at an endogenous immunoglobulin locus. The vagaries of such transgenic mice render them less than optimal for generating a sufficiently diverse human antibody repertoire in mice, likely due at least in part to a suboptimal clonal selection process that interfaces fully human antibody molecules within an endogenous selection system and deleterious effects from changes to the endogenous genetic makeup of such mice.

[0008] There remains a need in the art for making improved genetically modified non- human animals that are useful in generating immunoglobulin sequences, including human antibody sequences, and that are useful in generating a diverse repertoire of immunoglobulin-like molecules that exhibit diversity apart from traditional antibody molecules, while at the same time reducing or eliminating deleterious changes that might result from the genetic modifications. There also remains a need for non-human animals that are capable of rearranging immunoglobulin gene segments to form useful rearranged immunoglobulin genes, including human immunoglobulin light chain variable domains in the context of heavy chain constant domains that are cognate with human immunoglobulin light chain variable domains in the context of light chain constant domains, or that are capable of making proteins from altered immunoglobulin loci, including loci that contain a sufficiently diverse collection of human light chain variable gene segments. There remains a need for non-human animals that can generate immunoglobulin-like binding proteins, wherein the binding proteins comprise human immunoglobulin light chain variable domains linked to heavy chain constant domains. SUMMARY OF INVENTION

[0009] Genetically modified non-human animals having immunoglobulin loci are provided, wherein the immunoglobulin loci comprise a plurality of human light chain variable <com:Math><semantics>(VL)<annotation encoding="application / x-tex">(V_L)< / annotation>< / semantics>< / com:Math> gene segments operably linked to one or more non-human constant regions, e.g., human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> or human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J_\lambda< / annotation>< / semantics>< / com:Math>, and in various embodiments the loci lack a sequence that encodes an endogenous functional ADAM6 protein. The non-human animals include rodents, e.g., mice and rats.

[0010] Loci are provided that are capable of rearranging and forming a gene encoding a light chain variable domain that is derived from a rearrangement involving a human light chain <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> gene segment and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J_\lambda< / annotation>< / semantics>< / com:Math> gene segment and in various embodiments, additionally a D<com:Sub>H< / com:Sub> gene segment, wherein in various embodiments the loci lack an endogenous functional ADAM6 gene or functional fragment thereof.

[0011] Modified immunoglobulin loci include loci that lack an endogenous functional ADAM6 gene and comprise human immunoglobulin sequences are provided, e.g., a human V<com:Sub>L< / com:Sub> segment operably linked to a human or (or human / non-human chimeric) non-human immunoglobulin constant sequence (and in operable linkage with, <com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, a V and / or a J segment). Modified loci that comprise multiple V<com:Sub>L< / com:Sub> gene segments and an ectopic nucleotide sequence that encodes an ADAM6 protein or fragment thereof that is functional in the non- human animal, are provided. Modified loci that comprise multiple V<com:Sub>L< / com:Sub> gene segments, operably linked with one or more <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> segments and / or one or more <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> segments, operably linked to a non-human immunoglobulin constant sequence, e.g., a rodent (e.g., mouse or rat) or human sequence, are provided. Non-human animals comprising such humanized loci are also provided, wherein the non-human animals exhibit wild-type fertility.

[0012] Non-human animals that comprise an immunoglobulin heavy chain variable locus (e.g., on a transgene or as an insertion or replacement at an endogenous non-human animal heavy chain variable locus) that comprises a plurality of human V<com:Sub>⊥< / com:Sub> gene segments operably linked to a human D and / or human J gene segment are provided. In various embodiments, the plurality of human V<com:Sub>L< / com:Sub> gene segments are operably linked to one or more human D and / or one or more human J gene segments at the endogenous immunoglobulin heavy chain variable gene locus of the non-human animal. In various embodiments, the non-human animals further comprise an ectopic nucleotide sequence that encodes an ADAM6 protein or homolog or ortholog thereof that is functional in the male non-human animal that comprises the modified heavy chain locus. In various embodiments, the ectopic nucleotide sequence is contiguous with at least one human V<com:Sub>L< / com:Sub> segment, a D<com:Sub>H< / com:Sub> gene segment, or a J<com:Sub>L< / com:Sub> gene segment. In various embodiments, the ectopic nucleotide sequence is contiguous with a non-immunoglobulin sequence in the genome of the non-human animal. In one embodiment, the ectopic nucleotide sequence is on the same chromosome as the modified heavy chain locus. In one embodiment, the ectopic nucleotide sequence is on a different chromosome as the modified heavy chain locus.

[0013] Non-human animals are provided that are modified at their immunoglobulin heavy chain variable region loci to delete all or substantially all (e.g., all functional segments, or nearly all functional segments) endogenous immunoglobulin V<com:Sub>H< / com:Sub> segments and that comprise a plurality of human V<com:Sub>L< / com:Sub> gene segments operably linked to a D<com:Sub>H< / com:Sub> and J segment or a J<com:Sub>L< / com:Sub> gene segment at the endogenous immunoglobulin heavy chain variable region locus of the non-human animal. Non-human animals comprising such loci and that lack an endogenous ADAM6 gene are also provided.

[0014] Methods are provided for making human immunoglobulin sequences in non- human animals. In various embodiments, the human immunoglobulin sequences are derived from a repertoire of immunoglobulin heavy chain sequences that comprise human V<com:Sub>L< / com:Sub> gene segments rearranged and in operable linkage with immunoglobulin heavy chain constant regions, e.g., V<com:Sub>L< / com:Sub>, and one or more D<com:Sub>H< / com:Sub> and J segments or one or more J<com:Sub>L< / com:Sub> segments. Methods for making human immunoglobulin sequences in non-human animals, tissues, and cells are provided, wherein the human immunoglobulin sequences bind an antigen of interest.

[0015] In one aspect, nucleic acid constructs, cells, embryos, rodents (e.g., mice), and methods are provided for making rodents (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, mice) that comprise a modification that results in a nonfunctional endogenous rodent (e.g., mouse) ADAM6 protein or ADAM6 gene (e.g., a knockout of or a deletion in an endogenous ADAM6 gene), wherein the rodents (e.g., mice) comprise a nucleic acid sequence that encodes an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male rodent of the same kind (e.g., mouse). In one embodiment, the mice comprise an ectopic nucleotide sequence encoding a rodent ADAM6 protein or ortholog or homolog or functional fragment thereof; in a specific embodiment, the rodent ADAM6 protein is a mouse ADAM6 protein. In one embodiment, the mice comprise an ectopic nucleotide sequence that encodes one or more rodent ADAM6 proteins, wherein the one or more proteins comprise SEQ ID NO: 1 or SEQ ID NO: 2 or a fragment thereof that is functional in the mice.

[0016] In various aspects, the sequence that encodes ADAM6 activity is contiguous with a human immunoglobulin sequence. In various aspects, the sequence that encodes ADAM6 activity is contiguous with a non-human immunoglobulin sequence. In various aspects, the sequence is present on the same chromosome as the endogenous non-human immunoglobulin heavy chain locus of the non-human animal. In various aspects, the sequence is present on a different chromosome than the immunoglobulin heavy chain locus of the non-human animal.

[0017] Genetically modified non-human animals are described that comprise a modification that maintains activity of an ADAM6 gene or homolog or ortholog thereof, wherein the modification includes insertion of one or more human immunoglobulin light chain gene segments upstream of a non-human immunoglobulin heavy chain constant region, and the non-human animals further comprise modifications that enable them to express human immunoglobulin light chain variable regions cognate with human immunoglobulin light chain variable regions. In various aspects, the human immunoglobulin light chain variable regions are expressed in the context of light and heavy chain constant regions.

[0018] In various aspects, the insertion of one or more human immunoglobulin light chain gene segments is performed 3' or downstream of the ADAM6 gene of the non-human animal. In various aspects, the insertion of one or more human immunoglobulin light chain gene segments is performed in a manner such that the ADAM6 gene(s) of the non-human animal is not disrupted, deleted and / or functionally silenced such that the ADAM6 activity of the non-human animal is at the same or comparable level as in a non-human animal that does not contain such an insertion. Exemplary disruptions, deletions and / or functionally silencing modifications include any modification that results in a reduction, elimination and / or loss of activity of the ADAM6 protein(s) encoded by the ADAM6 gene(s) of the non-human animal.

[0019] In one aspect, nucleic acid constructs, cells, embryos, mice, and methods are provided for making mice that comprise a modification of an endogenous immunoglobulin locus, wherein the mice comprise an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the endogenous immunoglobulin locus is an immunoglobulin heavy chain locus, and the modification reduces or eliminates ADAM6 activity of a cell or tissue of a male mouse.

[0020] In one aspect, mice are provided that comprise an ectopic nucleotide sequence encoding a mouse ADAM6 or ortholog or homolog or functional fragment thereof; mice are also provided that comprise an endogenous nucleotide sequence encoding a mouse ADAM6 or ortholog or homolog or fragment thereof, and at least one genetic modification of a heavy chain immunoglobulin locus. In one embodiment, the endogenous nucleotide sequence encoding a mouse ADAM6 or ortholog or homolog or functional fragment thereof is located at an ectopic position as compared to an endogenous ADAM6 gene of a wild type mouse.

[0021] In one aspect, methods are provided for making mice that comprise a modification of an endogenous immunoglobulin locus, wherein the mice comprise an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse. In various embodiments, the modification comprises an insertion of one or more human V<com:Sub>L< / com:Sub> gene segments at the endogenous immunoglobulin locus.

[0022] In one aspect, methods are provided for making mice that comprise a genetic modification of a heavy chain immunoglobulin locus, wherein application of the methods result in male mice that comprise a modified heavy chain immunoglobulin locus (or a deletion thereof), and the male mice are capable of generating offspring by mating. In one embodiment, the male mice are capable of producing sperm that can transit from a mouse uterus through a mouse oviduct to fertilize a mouse egg.

[0023] In one aspect, methods are provided for making mice that comprise a genetic modification of an immunoglobulin heavy chain locus and an immunoglobulin light chain locus, wherein application of the methods to modify the heavy chain locus result in male mice that exhibit a reduction in fertility, and the mice comprise a genetic modification that restores in whole or in part the reduction in fertility. In various embodiments, the reduction in fertility is characterized by an inability of the sperm of the male mice to migrate from a mouse uterus through a mouse oviduct to fertilize a mouse egg. In various embodiments, the reduction in fertility is characterized by sperm that exhibit an in vivo migration defect. In various embodiments, the genetic modification that restores in whole or in part the reduction in fertility is a nucleic acid sequence encoding a mouse ADAM6 gene or ortholog or homolog or fragment thereof that is functional in a male mouse.

[0024] In one embodiment, the genetic modification comprises replacing endogenous immunoglobulin heavy chain variable loci with immunoglobulin light chain variable loci of another species (e.g., a non-mouse species). In one embodiment, the genetic modification comprises insertion of immunoglobulin light chain variable loci of another species (e.g., a non-mouse species) into endogenous immunoglobulin heavy chain variable loci. In a specific embodiment, the species is human. In one embodiment, the genetic modification comprises deletion of an endogenous immunoglobulin heavy chain variable locus in whole or in part, wherein the deletion results in a loss of endogenous ADAM6 function. In a specific embodiment, the loss of endogenous ADAM6 function is associated with a reduction in fertility in male mice.

[0025] In one embodiment, the genetic modification comprises inactivation of an endogenous non-human immunoglobulin heavy chain variable locus in whole or in part, wherein the inactivation does not result in a loss of endogenous ADAM6 function. Inactivation may include replacement or deletion of one or more endogenous non-human gene segments resulting in an endogenous non-human immunoglobulin heavy chain locus that is substantially incapable of rearrangement to encode a heavy chain of an antibody that comprises endogenous non-human gene segments. Inactivation may include other modifications that render the endogenous immunoglobulin heavy chain locus incapable of rearranging to encode the heavy chain of an antibody, wherein the modification does not include replacement or deletion of endogenous gene segments. Exemplary modifications include chromosomal inversions and / or translocations mediated by molecular techniques, e.g., using precise placement of site-specific recombination sites (e.g., Cre-lox technology). Other exemplary modifications include disabling the operable linkage between the non- human immunoglobulin variable gene segments and the non-human immunoglobulin constant regions.

[0026] In one embodiment, the genetic modification comprises inserting into the genome of the non-human animal a DNA fragment containing one or more human V<com:Sub>L< / com:Sub> gene segments and one or more human J<com:Sub>L< / com:Sub> gene segments, and optionally one or more human D<com:Sub>H< / com:Sub> gene segments, of another species (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, a non-mouse species) operably linked to one or more constant region sequences (e.g., an IgM and / or an IgG gene). In one embodiment, the DNA fragment is capable of undergoing rearrangement in the genome of the non-human animal to form a sequence that encodes a light chain variable domain operably linked to a heavy chain constant region. In one embodiment, the species is human. In one embodiment, the genetic modification comprises insertion of one or more human immunoglobulin light chain gene segments downstream or 3' of an endogenous ADAM6 gene of the non-human animal such that ADAM6 activity (e.g., expression and / or function of an encoded protein) is the same or comparable to a non-human animal that does not comprise the insertion.

[0027] In one aspect, methods are provided for making mice that comprise a genetic modification of an immunoglobulin heavy chain locus, wherein application of the methods result in male mice that comprise a modified immunoglobulin heavy chain locus (or a deletion thereof), and the male mice exhibit a reduction in fertility, and the mice comprise a genetic modification that restores in whole or in part the reduction in fertility. In various embodiments, the reduction in fertility is characterized by an inability of the sperm of the male mice to migrate from a mouse uterus through a mouse oviduct to fertilize a mouse egg. In various embodiments, the reduction in fertility is characterized by sperm that exhibit an in vivo migration defect. In various embodiments, the genetic modification that restores in whole or in part the reduction in fertility is a nucleic acid sequence encoding a mouse ADAM6 gene or ortholog or homolog or fragment thereof that is functional in a male mouse.

[0028] In one embodiment, the genetic modification comprises replacing endogenous immunoglobulin heavy chain variable loci with immunoglobulin light chain variable loci, e.g., one or more light chain variable <com:Math><semantics>(VL)<annotation encoding="application / x-tex">(V_L)< / annotation>< / semantics>< / com:Math> gene segments, one or more heavy chain diversity <com:Math><semantics>(DH)<annotation encoding="application / x-tex">(D_H)< / annotation>< / semantics>< / com:Math> gene segments and one or more joining (J) gene segments, or one or more light chain joining <com:Math><semantics>(JL)<annotation encoding="application / x-tex">(J_L)< / annotation>< / semantics>< / com:Math> gene segments of another species <com:Math><semantics>(e.g.,anon−mousespecies)<annotation encoding="application / x-tex">(e.g., a non-mouse species)< / annotation>< / semantics>< / com:Math>. In one embodiment, the genetic modification comprises insertion of a single orthologous immunoglobulin light chain variable loci V<com:Sub>L< / com:Sub> gene segment, at least one D<com:Sub>H< / com:Sub> gene segment and at least one J gene segment, or at least one J<com:Sub>L< / com:Sub> gene segment into endogenous immunoglobulin heavy chain variable loci. In a specific embodiment, the species is human. In one embodiment, the genetic modification comprises a deletion of an endogenous immunoglobulin heavy chain variable locus in whole or in part, wherein the deletion results in a loss of endogenous ADAM6 function. In a specific embodiment, the loss of endogenous ADAM6 function is associated with a reduction in fertility in male mice. In one embodiment, the genetic modification comprises inactivation of an endogenous immunoglobulin heavy chain variable locus in whole or in part, wherein the deletion does not result in a loss of endogenous ADAM6 function. Inactivation may include replacement or deletion of one or more endogenous gene segments resulting in an endogenous immunoglobulin heavy chain locus that is substantially incapable of rearrangement to encode a heavy chain of an antibody that comprises endogenous gene segments. Inactivation may include other modifications that render the endogenous immunoglobulin heavy chain locus incapable of rearranging to encode the heavy chain of an antibody, wherein the modification does not include replacement or deletion of endogenous gene segments. Exemplary modifications include chromosomal inversions and / or alterations that result in a heavy chain locus that is not in operable linkage with one or more endogenous constant regions.

[0029] In one embodiment, the genetic modification comprises inserting into the genome of the mouse a DNA fragment containing one or more human V<com:Sub>L< / com:Sub> gene segments, one or more J gene segments, and optionally one or more D gene segments of another species (e.g., a non-mouse species) operably linked to one or more constant region sequences (e.g., an IgM and / or an IgG gene). In various embodiments, the J gene segments include <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the DNA fragment is capable of undergoing rearrangement to form a sequence that encodes a heavy chain of an antibody, wherein the heavy chain comprises a rearranged human light chain variable gene segment fused to a heavy chain constant region. In one embodiment, the genetic modification comprises insertion of at least six, at least 16, at least 30, or at least 40 or more human V<com:Sub>L< / com:Sub> gene segments, and at least one or at least 5 human J<com:Sub>L< / com:Sub> gene segments into the genome of the mouse. In a specific embodiment, the species is human and the gene segments are human κ light chain gene segments. In one embodiment, the genetic modification comprises deletion of an endogenous immunoglobulin heavy chain variable locus in whole or in part to render the endogenous immunoglobulin heavy chain locus nonfunctional, wherein the deletion further results in a loss of endogenous ADAM6 function. In a specific embodiment, the loss of endogenous ADAM6 function is associated with a reduction in fertility in male mice.

[0030] In one aspect, mice are provided that comprise a modification that reduces or eliminates mouse ADAM6 expression from an endogenous ADAM6 allele such that a male mouse having the modification exhibits a reduced fertility (e.g., a highly reduced ability to generate offspring by mating), or is essentially infertile, due to the reduction or elimination of endogenous ADAM6 function, wherein the mice further comprise an ectopic ADAM6 sequence or homolog or ortholog or functional fragment thereof. In one aspect, the modification that reduces or eliminates mouse ADAM6 expression is a modification (e.g., an insertion, a deletion, a replacement, etc.) in a mouse immunoglobulin locus. In one embodiment, the immunoglobulin locus is an immunoglobulin heavy chain locus.

[0031] In one embodiment, the reduction or loss of ADAM6 function comprises an inability or substantial inability of the mouse to produce sperm that can travel from a mouse uterus through a mouse oviduct to fertilize a mouse egg. In a specific embodiment, at least about 95%, 96%, 97%, 98%, or 99% of the sperm cells produced in an ejaculate volume of the mouse are incapable of traversing through an oviduct in vivo following copulation and fertilizing a mouse ovum.

[0032] In one embodiment, the reduction or loss of ADAM6 function comprises an inability to form or substantial inability to form a complex of ADAM2 and / or ADAM3 and / or ADAM6 on a surface of a sperm cell of the mouse. In one embodiment, the loss of ADAM6 function comprises a substantial inability to fertilize a mouse egg by copulation with a female mouse.

[0033] In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 gene, and comprises a protein (or an ectopic nucleotide sequence that encodes a protein) that confers ADAM6 functionality on the mouse. In one embodiment, the mouse is a male mouse and the functionality comprises enhanced fertility as compared with a mouse that lacks a functional endogenous ADAM6 gene.

[0034] In one embodiment, the protein is encoded by a genomic sequence located within an immunoglobulin locus in the germline of the mouse. In a specific embodiment, the immunoglobulin locus is a heavy chain locus. In another specific embodiment, the heavy chain locus comprises at least one human V<com:Sub>H< / com:Sub>, at least one human D<com:Sub>H< / com:Sub> and at least one human J<com:Sub>H< / com:Sub> gene segment. In another specific embodiment, the heavy chain locus comprises at least one human V<com:Sub>L< / com:Sub> and at least one human J<com:Sub>L< / com:Sub> gene segment. In another specific embodiment, the heavy chain locus comprises at least one human V<com:Sub>L< / com:Sub>, at least one human <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math>, and at least one human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math>. In another specific embodiment, the heavy chain locus comprises at least one human V<com:Sub>L< / com:Sub>, at least one human D<com:Sub>H< / com:Sub>, and at least one human J<com:Sub>H< / com:Sub> gene segment. In another specific embodiment, the heavy chain locus comprises at least one human V<com:Sub>L< / com:Sub> and at least one human J<com:Sub>L< / com:Sub> gene segment. In another specific embodiment, the heavy chain locus comprises at least one human V<com:Sub>L< / com:Sub> and at least one human J<com:Sub>H< / com:Sub> gene segment. In another specific embodiment, the heavy chain locus comprises six human V<com:Sub>K< / com:Sub> and five human Jk gene segments. In another specific embodiment, the heavy chain locus comprises 16 human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and five human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In another specific embodiment, the heavy chain locus comprises 30 human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and five human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In another specific embodiment, the heavy chain locus comprises 40 human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> and five human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments.

[0035] In one embodiment, the ectopic protein is encoded by a genomic sequence located within a non-immunoglobulin locus in the germline of the mouse. In one embodiment, the non-immunoglobulin locus is a transcriptionally active locus. In a specific embodiment, the transcriptionally active locus is the ROSA locus. In a specific embodiment, the transcriptionally active locus is associated with tissue-specific expression. In one embodiment, the tissue-specific expression is present in reproductive tissues. In one embodiment, the protein is encoded by a genomic sequence randomly inserted into the germline of the mouse.

[0036] In one embodiment, the mouse comprises a human or chimeric human / mouse or chimeric human / rat light chain (e.g., human variable, mouse or rat constant) and a chimeric human variable / mouse or rat constant heavy chain. In a specific embodiment, the mouse comprises a transgene that comprises a chimeric human variable / rat or mouse constant light chain gene operably linked to a transcriptionally active promoter, e.g., a ROSA26 promoter. In a further specific embodiment, the chimeric human / mouse or rat light chain transgene comprises a rearranged human light chain variable region sequence in the germline of the mouse.

[0037] In one embodiment, the ectopic nucleotide sequence is located within an immunoglobulin locus in the germline of the mouse. In a specific embodiment, the immunoglobulin locus is a heavy chain locus. In one embodiment, the heavy chain locus comprises at least one human V<com:Sub>L< / com:Sub> and at least one human J<com:Sub>L< / com:Sub> gene segment. In a specific embodiment, the heavy chain locus comprises at least six and up to 40 human Vκ gene segments, and five human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the ectopic nucleotide sequence is located within a non-immunoglobulin locus in the germline of the mouse. In one embodiment, the non-immunoglobulin locus is a transcriptionally active locus. In a specific embodiment, the transcriptionally active locus is the ROSA26 locus. In one embodiment, the ectopic nucleotide sequence is positioned randomly inserted into the germline of the mouse.

[0038] In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 gene, wherein the mouse comprises an ectopic nucleotide sequence that complements the loss of mouse ADAM6 function. In one embodiment, the ectopic nucleotide sequence confers upon the mouse an ability to produce offspring that is comparable to a corresponding wild-type mouse that contains a functional endogenous ADAM6 gene. In one embodiment, the sequence confers upon the mouse an ability to form a complex of ADAM2 and / or ADAM3 and / or ADAM6 on the surface of sperm cell of the mouse. In one embodiment, the sequence confers upon the mouse an ability to travel from a mouse uterus through a mouse oviduct to a mouse ovum to fertilize the ovum.

[0039] In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces at least about 50%, 60%, 70%, 80%, or 90% of the number of litters a wild-type mouse of the same age and strain produces in a six-month time period.

[0040] In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces at least about 1.5-fold, about 2- fold, about 2.5-fold, about 3-fold, about 4-fold, about 6-fold, about 7-fold, about 8-fold, or about 10-fold or more progeny when bred over a six-month time period than a mouse of the same age and the same or similar strain that lacks the functional endogenous ADAM6 gene and that lacks the ectopic nucleotide sequence that is bred over substantially the same time period and under substantially the same conditions.

[0041] In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces an average of at least about 2- fold, 3-fold, or 4-fold higher number of pups per litter in a four or six month breeding period than a mouse that lacks the functional endogenous ADAM6 gene and that lacks the ectopic nucleotide sequence, and that is bred for the same period of time.

[0042] In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence is a male mouse, and the male mouse produces sperm that when recovered from oviducts at about 5-6 hours post-copulation reflects an oviduct migration that is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, 100-fold, 110-fold, or 120-fold or higher than a mouse that lacks the functional endogenous ADAM6 gene and that lacks the ectopic nucleotide sequence.

[0043] In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence when copulated with a female mouse generates sperm that is capable of traversing the uterus and entering and traversing the oviduct within about six hours at an efficiency that is about equal to sperm from a wild-type mouse.

[0044] In one embodiment, the mouse lacking the functional endogenous ADAM6 gene and comprising the ectopic nucleotide sequence produces about 1.5-fold, about 2-fold, about 3-fold, or about 4-fold or more litters in a comparable period of time than a mouse that lacks the functional ADAM6 gene and that lacks the ectopic nucleotide sequence.

[0045] In one aspect, a mouse comprising in its germline a non-mouse nucleic acid sequence that encodes an immunoglobulin protein is provided, wherein the non-mouse immunoglobulin sequence comprises an insertion of a mouse ADAM6 gene or homolog or ortholog or functional fragment thereof. In one embodiment, the non-mouse immunoglobulin sequence comprises a human immunoglobulin sequence. In one embodiment, the sequence comprises a human immunoglobulin heavy chain sequence. In one embodiment, the sequence comprises a human immunoglobulin light chain sequence. In one embodiment, the sequence comprises a human heavy chain sequence contiguous with a human light chain sequence. In one embodiment, the sequence comprises one or more V gene segments, one or more D gene segments, and one or more J gene segments; in one embodiment, the sequence comprises one or more V gene segments and one or more J gene segments. In one embodiment, the one or more V, D, and J gene segments, or one or more V and J gene segments, are unrearranged. In one embodiment, the one or more V, D, and J gene segments, or one or more V and J gene segments, are rearranged. In one embodiment, following rearrangement of the one or more V, D, and J gene segments, or one or more V and J gene segments, the mouse comprises in its genome at least one nucleic acid sequence encoding a mouse ADAM6 gene or homolog or ortholog or functional fragment thereof. In one embodiment, following rearrangement the mouse comprises in its genome at least two nucleic acid sequences encoding a mouse ADAM6 gene or homolog or ortholog or functional fragment thereof. In one embodiment, following rearrangement the mouse comprises in its genome at least one nucleic acid sequence encoding a mouse ADAM6 gene or homolog or ortholog or functional fragment thereof. In one embodiment, the mouse comprises the ADAM6 gene or homolog or ortholog or functional fragment thereof in a B cell. In one embodiment, the mouse comprises the ADAM6 gene or homolog or ortholog or functional fragment thereof in a non-B cell.

[0046] In one aspect, mice are provided that express a human immunoglobulin heavy chain variable region or functional fragment thereof from an endogenous immunoglobulin heavy chain locus, wherein the mice comprise an ADAM6 activity that is functional in a male mouse. In one embodiment, the heavy chain locus comprises one or more human V<com:Sub>L< / com:Sub> gene segments and one or more J<com:Sub>L< / com:Sub> gene segments, and optionally one or more D<com:Sub>H< / com:Sub> gene segments. In one embodiment, the heavy chain locus comprises at least six human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> gene segments and five human <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the heavy chain locus comprises at least 16 human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments and five human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the heavy chain locus comprises at least 30 human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments and five human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the heavy chain locus comprises at least 40 human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> gene segments and five human <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J_{\kappa}< / annotation>< / semantics>< / com:Math> gene segments.

[0047] In one aspect, mice are provided that express a human immunoglobulin light chain variable region or functional fragment thereof from an endogenous immunoglobulin heavy chain locus, wherein the mice comprise an ADAM6 activity that is functional in a male mouse.

[0048] In one embodiment, the male mice comprise a single unmodified endogenous ADAM6 allele or ortholog of homolog or functional fragment thereof at an endogenous ADAM6 locus.

[0049] In one embodiment, the male mice comprise an ectopic mouse ADAM6 sequence or homolog or ortholog or functional fragment thereof that encodes a protein that confers ADAM6 function.

[0050] In one embodiment, the male mice comprise an ADAM6 sequence or homolog or ortholog or functional fragment thereof at a location in the mouse genome that approximates the location of the endogenous ADAM6 allele, e.g., 3' of a final V gene segment sequence and 5' of an initial J gene segment.

[0051] In one embodiment, the male mice comprise an ADAM6 sequence or homolog or ortholog or functional fragment thereof at a location in the mouse genome that is different from that of the endogenous ADAM6 allele, e.g., 5' of the 5'-most V gene segment sequence of a V gene locus.

[0052] In one embodiment, the male mice comprise an ADAM6 sequence or homolog or ortholog or functional fragment thereof flanked upstream, downstream, or upstream and downstream (with respect to the direction of transcription of the ADAM6 sequence) of a nucleic acid sequence encoding an immunoglobulin V gene segment and / or an immunoglobulin J gene segment. In a specific embodiment, the immunoglobulin V and J gene segments are human gene segments. In one embodiment, the immunoglobulin V and J gene segments are human gene segments, and the sequence encoding the mouse ADAM6 or ortholog or homolog or fragment thereof functional in a mouse is between the human V and J gene segments; in one embodiment, the mouse comprises two or more human V gene segments, and the sequence is at a position 5' of the 5'-most human V gene segment; in one embodiment, the mouse comprises two or more human V gene segments, and the sequence is at a position between the final V gene segment and the penultimate V gene segment; in one embodiment, the mouse comprises a plurality of human V gene segments, and the sequence is at a position upstream of the 5'-most human V gene segment; in one embodiment, the mouse further comprises a D gene segment, and the sequence is at a position following the 3'-most V gene segment and a 5'-most D gene segment; in one embodiment, the sequence is at a position between a V gene segment and a J gene segment.

[0053] In one embodiment, the human V gene segments are light chain V gene segments. In a specific embodiment, the light chain V gene segments are Vκ gene segments. In another specific embodiment, the light chain V gene segments are <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the J gene segment is selected from a J<com:Sub>H< / com:Sub> and a J<com:Sub>L< / com:Sub> gene segment. In a specific embodiment, the <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segment is a <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment. In another specific embodiment, the <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segment is a <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segment.

[0054] In one embodiment, the male mice comprise an ADAM6 sequence or homolog or ortholog or functional fragment thereof that is located at a position in an endogenous immunoglobulin locus that is the same or substantially the same as in a wild type male mouse. In a specific embodiment, the endogenous locus is incapable of encoding the heavy chain variable region of an antibody, wherein the variable region comprises or is derived from an endogenous non-human gene segment. In a specific embodiment, the endogenous locus is positioned at a location in the genome of the male mouse that renders the heavy chain gene segments of the locus incapable of encoding a heavy chain variable region of an antibody. In various embodiments, the male mice comprise an ADAM6 sequence located on the same chromosome as human immunoglobulin gene segments and the ADAM6 sequence encodes a functional ADAM6 protein.

[0055] In one aspect, a male mouse is provided that comprises a nonfunctional endogenous ADAM6 gene, or a deletion of an endogenous ADAM6 gene, in its germline; wherein sperm cells of the mouse are capable of transiting an oviduct of a female mouse and fertilizing an egg. In one embodiment, the mice comprise an extrachromosomal copy of a mouse ADAM6 gene or ortholog or homolog or functional fragment thereof that is functional in a male mouse. In one embodiment, the mice comprise an ectopic mouse ADAM6 gene or ortholog or homolog or functional fragment thereof that is functional in a male mouse.

[0056] In one aspect, a male mouse is provided that comprises a functional endogenous ADAM6 gene and a modification to an endogenous immunoglobulin heavy chain locus. In one embodiment, the modification is made downstream, or 3' of an endogenous ADAM6 gene or locus. In one embodiment, the modification is a replacement of one or more endogenous immunoglobulin heavy chain gene segments with one or more human immunoglobulin light chain gene segments. In one embodiment, the modification is an insertion of one or more human immunoglobulin light chain gene segments upstream of an endogenous immunoglobulin heavy chain constant region gene.

[0057] In one aspect, mice are provided that comprise a genetic modification that reduces endogenous ADAM6 function, wherein the mouse comprises at least some ADAM6 functionality provided either by an endogenous unmodified allele that is functional in whole or in part (e.g., a heterozygote), or by expression from an ectopic sequence that encodes an ADAM6 or an ortholog or homolog or functional fragment thereof that is functional in a male mouse. In various embodiments, the ADAM6 or ortholog or homolog or functional fragment thereof comprises a nucleic acid sequence that encodes an ADAM6 protein set forth in SEQ ID NO: 1, SEQ ID NO: 2, or a combination thereof.

[0058] In one embodiment, the mice comprise ADAM6 function sufficient to confer upon male mice the ability to generate offspring by mating, as compared with male mice that lack a functional ADAM6. In one embodiment, the ADAM6 function is conferred by the presence of an ectopic nucleotide sequence that encodes a mouse ADAM6 or a homolog or ortholog or functional fragment thereof. In one embodiment, the ADAM6 function is conferred by an endogenous ADAM6 gene present in an endogenous immunoglobulin locus, wherein the mouse is incapable of expressing an antibody comprising an endogenous immunoglobulin heavy chain gene segment. ADAM6 homologs or orthologs or fragments thereof that are functional in a male mouse include those that restore, in whole or in part, the loss of ability to generate offspring observed in a male mouse that lacks sufficient endogenous ADAM6 activity, e.g., the loss in ability observed in an ADAM6 knockout mouse. In this sense ADAM6 knockout mice include mice that comprise an endogenous locus or fragment thereof, but that is not functional, i.e., that does not express ADAM6 (ADAM6a and / or ADAM6b) at all, or that expresses ADAM6 (ADAM6a and / or ADAM6b) at a level that is insufficient to support an essentially normal ability to generate offspring of a wild-type male mouse. The loss of function can be due, e.g., to a modification in a structural gene of the locus (i.e., in an ADAM6a or ADAM6b coding region) or in a regulatory region of the locus (e.g., in a sequence 5' to the ADAM6a gene, or 3' of the ADAM6a or ADAM6b coding region, wherein the sequence controls, in whole or in part, transcription of an ADAM6 gene, expression of an ADAM6 RNA, or expression of an ADAM6 protein). In various embodiments, orthologs or homologs or fragments thereof that are functional in a male mouse are those that enable a sperm of a male mouse (or a majority of sperm cells in the ejaculate of a male mouse) to transit a mouse oviduct and fertilize a mouse ovum.

[0059] In one embodiment, male mice that express the human immunoglobulin variable region or functional fragment thereof comprise sufficient ADAM6 activity to confer upon the male mice the ability to generate offspring by mating with female mice and, in one embodiment, the male mice exhibit an ability to generate offspring when mating with female mice that is in one embodiment at least 25%, in one embodiment, at least 30%, in one embodiment at least 40%, in one embodiment at least 50%, in one embodiment at least 60%, in one embodiment at least 70%, in one embodiment at least 80%, in one embodiment at least 90%, and in one embodiment about the same as, that of mice with one or two endogenous unmodified ADAM6 alleles.

[0060] In one embodiment male mice express sufficient ADAM6 (or an ortholog or homolog or functional fragment thereof) to enable a sperm cell from the male mice to traverse a female mouse oviduct and fertilize a mouse egg.

[0061] In one embodiment, the ADAM6 functionality is conferred by a nucleic acid sequence that is contiguous with a mouse chromosomal sequence (e.g., the nucleic acid is randomly integrated into a mouse chromosome; or placed at a specific location, e.g., by targeting the nucleic acid to a specific location, e.g., by site-specific recombinase-mediated (e.g., Cre-mediated) insertion or homologous recombination). In one embodiment, the ADAM6 sequence is present on a nucleic acid that is distinct from a chromosome of the mouse (e.g., the ADAM6 sequence is present on an episome, i.e., extra-chromosomally, e.g., in an expression construct, a vector, a YAC, a trans-chromosome, etc.).

[0062] In one aspect, genetically modified mice and cells are provided that comprise a modification of an endogenous heavy chain immunoglobulin locus, wherein the mice express at least a portion of a immunoglobulin light chain sequence, e.g., at least a portion of a human sequence, wherein the mice comprise an ADAM6 activity that is functional in a male mouse. In one embodiment, the modification reduces or eradicates an ADAM6 activity of the mouse. In one embodiment, the mouse is modified such that both alleles that encode ADAM6 activity are either absent or express an ADAM6 that does not substantially function to support normal mating in a male mouse. In one embodiment, the mouse further comprises an ectopic nucleic acid sequence encoding a mouse ADAM6 or ortholog or homolog or functional fragment thereof. In one embodiment, the modification maintains ADAM6 activity of the mouse and renders an endogenous immunoglobulin heavy chain locus incapable of encoding a heavy chain of an antibody. In a specific embodiment, the modification includes chromosomal inversions and or translocations that render the endogenous immunoglobulin heavy chain locus incapable of rearranging to encode a heavy chain variable region of an antibody.

[0063] In one aspect, genetically modified mice and cells are provided that comprise a modification of an endogenous immunoglobulin heavy chain locus, wherein the modification reduces or eliminates ADAM6 activity expressed from an ADAM6 sequence of the locus, and wherein the mice comprise an ADAM6 protein or ortholog or homolog or functional fragment thereof. In various embodiments, the ADAM6 protein or fragment thereof is encoded by an ectopic ADAM6 sequence. In various embodiments, the ADAM6 protein or fragment thereof is expressed from an endogenous ADAM6 allele. In various embodiments, the mouse comprises a first heavy chain allele comprises a first modification that reduces or eliminates expression of a functional ADAM6 from the first heavy chain allele, and the mouse comprises a second heavy chain allele that comprises a second modification that does not substantially reduce or does not eliminate expression of a functional ADAM6 from the second heavy chain allele.

[0064] In various embodiments, the modification is the insertion of one or more human immunoglobulin light chain gene segments upstream, or 5', of an endogenous immunoglobulin heavy chain constant region gene. In various embodiments, the modification maintains the endogenous ADAM6 gene located at the endogenous immunoglobulin heavy chain locus.

[0065] In one embodiment, the second modification is located 3' (with respect to the transcriptional directionality of the mouse V gene segment) of a final mouse V gene segment and located 5' (with respect to the transcriptional directionality of the constant sequence) of a mouse (or chimeric human / mouse) immunoglobulin heavy chain constant gene or fragment thereof (e.g., a nucleic acid sequence encoding a human and / or mouse: C<com:Sub>H< / com:Sub>1 and / or hinge and / or C<com:Sub>H< / com:Sub>2 and / or C<com:Sub>H< / com:Sub>3).

[0066] In one embodiment, the modification is at a first immunoglobulin heavy chain allele at a first locus that encodes a first ADAM6 allele, and the ADAM6 function results from expression of an endogenous ADAM6 at a second immunoglobulin heavy chain allele at a second locus that encodes a functional ADAM6, wherein the second immunoglobulin heavy chain allele comprises at least one modification of a V, D, and / or J gene segment. In a specific embodiment, the at least one modification of the V, D, and or J gene segment is a deletion, a replacement with a human V, D, and / or J gene segment, a replacement with a camelid V, D, and / or J gene segment, a replacement with a humanized or camelized V, D, and / or J gene segment, a replacement of a heavy chain sequence with a light chain sequence, and a combination thereof. In one embodiment, the at least one modification is the deletion of one or more heavy chain V, D, and / or J gene segments and a replacement with one or more light chain V and / or J gene segments (e.g., a human light chain V and / or J gene segment) at the heavy chain locus.

[0067] In one embodiment, the modification is at a first immunoglobulin heavy chain allele at a first locus and a second immunoglobulin heavy chain allele at a second locus, and the ADAM6 function results from expression of an ectopic ADAM6 at a non-immunoglobulin locus in the germline of the mouse. In a specific embodiment, the non-immunoglobulin locus is the ROSA26 locus. In a specific embodiment, the non-immunoglobulin locus is transcriptionally active in reproductive tissue.

[0068] In one embodiment, the modification is at a first immunoglobulin heavy chain allele at a first locus and a second immunoglobulin heavy chain allele at a second locus, and the ADAM6 function results from an endogenous ADAM6 gene in the germline of the mouse. In a specific embodiment, the endogenous ADAM6 gene is juxtaposed by mouse immunoglobulin heavy chain gene segments.

[0069] In one embodiment, the modification is at a first immunoglobulin heavy chain allele at a first locus and a second immunoglobulin heavy chain allele at a second locus, and the ADAM6 function results from expression of an ectopic ADAM6 sequence at the first immunoglobulin heavy chain allele. In one embodiment, the modification is at a first immunoglobulin heavy chain allele at a first locus and a second immunoglobulin heavy chain allele at a second locus, and the ADAM6 function or activity results from expression of an ectopic ADAM6 at the second immunoglobulin heavy chain allele.

[0070] In one aspect, a mouse comprising a heterozygous or a homozygous knockout of ADAM6 is provided. In one embodiment, the mouse further comprises a modified immunoglobulin sequence that is a human or a humanized immunoglobulin sequence, or a camelid or camelized human or mouse immunoglobulin sequence. In one embodiment, the modified immunoglobulin sequence is present at the endogenous immunoglobulin heavy chain locus. In one embodiment, the modified immunoglobulin sequence comprises a human light chain variable region sequence at an endogenous immunoglobulin heavy chain locus. In one embodiment, the human light chain variable region sequence replaces an endogenous heavy chain variable sequence at the endogenous immunoglobulin heavy chain locus. In one embodiment, the modified immunoglobulin sequence comprises a human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence at an endogenous immunoglobulin heavy chain locus. In one embodiment, the modified immunoglobulin sequence comprises a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence at an endogenous immunoglobulin heavy chain locus.

[0071] In one aspect, a mouse incapable of expressing a functional endogenous ADAM6 from an endogenous ADAM6 locus is provided. In one embodiment, the mouse comprises an ectopic nucleic acid sequence that encodes an ADAM6, or functional fragment thereof, that is functional in the mouse. In a specific embodiment, the ectopic nucleic acid sequence encodes a protein that rescues a loss in the ability to generate offspring exhibited by a male mouse that is homozygous for an ADAM6 knockout. In a specific embodiment, the ectopic nucleic acid sequence encodes a mouse ADAM6 protein.

[0072] In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 locus, and that comprises an ectopic nucleic acid sequence that confers upon the mouse ADAM6 function. In one embodiment, the nucleic acid sequence comprises an endogenous ADAM6 sequence or functional fragment thereof. In one embodiment, the endogenous ADAM6 sequence comprises ADAM6a- and ADAM6b-encoding sequence located in a wild- type mouse between the 3'-most mouse immunoglobulin heavy chain V gene segment (V<com:Sub>H< / com:Sub>) and the 5'-most mouse immunoglobulin heavy chain D gene segment (D<com:Sub>H< / com:Sub>).

[0073] In one embodiment, the nucleic acid sequence comprises a sequence encoding mouse ADAM6a or functional fragment thereof and / or a sequence encoding mouse ADAM6b or functional fragment thereof, wherein the ADAM6a and / or ADAM6b or functional fragment(s) thereof is operably linked to a promoter. In one embodiment, the promoter is a human promoter. In one embodiment, the promoter is the mouse ADAM6 promoter. In a specific embodiment, the ADAM6 promoter comprises sequence located between the first codon of the first ADAM6 gene closest to the mouse 5'-most D<com:Sub>H< / com:Sub> gene segment and the recombination signal sequence of the 5'-most D<com:Sub>H< / com:Sub> gene segment, wherein 5' is indicated with respect to direction of transcription of the mouse immunoglobulin genes. In one embodiment, the promoter is a viral promoter. In a specific embodiment, the viral promoter is a cytomegalovirus (CMV) promoter. In one embodiment, the promoter is an ubiquitin promoter.

[0074] In one embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter regulates expression in non-reproductive tissues. In one embodiment, the inducible promoter regulates expression in reproductive tissues. In a specific embodiment, the expression of the mouse ADAM6a and / or ADAM6b sequences or functional fragments(s) thereof is developmentally regulated by the inducible promoter in reproductive tissues.

[0075] In one embodiment, the mouse ADAM6a and / or ADAM6b are selected from the ADAM6a of SEQ ID NO: 1 and / or ADAM6b of sequence SEQ ID NO: 2.

[0076] In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO: 3. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO: 3 directly upstream (with respect to the direction of transcription of ADAM6a) of the first codon of ADAM6a and extending to the end of SEQ ID NO: 3 upstream of the ADAM6 coding region. In another specific embodiment, the ADAM6 promoter is a fragment extending from within about 5 to about 20 nucleotides upstream of the start codon of ADAM6a to about 0.5kb, 1kb, 2kb, or 3kb or more upstream of the start codon of ADAM6a.

[0077] In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 3 or a fragment thereof that when placed into a mouse that is infertile or that has low fertility due to a lack of ADAM6 improves fertility or restores fertility to about a wild-type fertility. In one embodiment, SEQ ID NO: 3 or a fragment thereof confers upon a male mouse the ability to produce a sperm cell that is capable of traversing a female mouse oviduct in order to fertilize a mouse egg.

[0078] In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO: 4. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO: 4 directly upstream (with respect to the direction of transcription of ADAM6a) of the first codon of ADAM6a and extending to the end of SEQ ID NO: 4 upstream of the ADAM6 coding region. In another specific embodiment, the ADAM6 promoter is a fragment extending from within about 5 to about 20 nucleotides upstream of the start codon of ADAM6a to about 0.5kb, 1kb, 2kb, or 3kb or more upstream of the start codon of ADAM6a.

[0079] In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 4 or a fragment thereof that when placed into a mouse that is infertile or that has low fertility due to a lack of ADAM6 improves fertility or restores fertility to about a wild-type fertility. In one embodiment, SEQ ID NO: 4 or a fragment thereof confers upon a male mouse the ability to produce a sperm cell that is capable of traversing a female mouse oviduct in order to fertilize a mouse egg.

[0800] In one embodiment, the mouse ADAM6 promoter is a promoter of SEQ ID NO: 5. In a specific embodiment, the mouse ADAM6 promoter comprises the nucleic acid sequence of SEQ ID NO: 5 directly upstream (with respect to the direction of transcription of ADAM6a) of the first codon of ADAM6a and extending to the end of SEQ ID NO: 5 upstream of the ADAM6 coding region. In another specific embodiment, the ADAM6 promoter is a fragment extending from within about 5 to about 20 nucleotides upstream of the start codon of ADAM6a to about 0.5kb, 1kb, 2kb, or 3kb or more upstream of the start codon of ADAM6a.

[0081] In one embodiment, the nucleic acid sequence comprises SEQ ID NO: 5 or a fragment thereof that when placed into a mouse that is infertile or that has low fertility due to a lack of ADAM6 improves fertility or restores fertility to about a wild-type fertility. In one embodiment, SEQ ID NO: 5 or a fragment thereof confers upon a male mouse the ability to produce a sperm cell that is capable of traversing a female mouse oviduct in order to fertilize a mouse egg.

[0082] In various embodiments, the ectopic nucleic acid sequence that confers upon the mouse ADAM6 function encodes one or more ADAM6 proteins, wherein the one or more ADAM6 proteins comprise SEQ ID NO: 1, SEQ ID NO: 2 or a combination thereof.

[0083] In various embodiments, the ectopic nucleic acid sequence comprises a sequence selected from SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, wherein the ectopic nucleic acid sequence confers upon the mouse ADAM6 function through one or more ADAM6 proteins encoded by the ectopic nucleic acid sequence.

[0084] In one embodiment, the nucleic acid sequence is any sequence encoding an ADAM6 gene or homolog or ortholog or functional fragment thereof that when placed into or maintained in a mouse yields a level of fertility that is the same or comparable to a wild-type mouse. An exemplary level of fertility may be demonstrated by the ability of a male mouse to produce a sperm cell that is capable of traversing a female mouse oviduct in order to fertilize a mouse egg.

[0085] In one aspect, a mouse is provided that comprises a deletion of an endogenous nucleotide sequence that encodes an ADAM6 protein, a replacement of an endogenous V<com:Sub>H< / com:Sub> gene segment with a human V<com:Sub>H< / com:Sub> gene segment, and an ectopic nucleotide sequence that encodes a mouse ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse.

[0086] In one aspect, a mouse is provided that comprises a deletion of an endogenous nucleotide sequence that encodes an ADAM6 protein, a replacement of an endogenous V<com:Sub>H< / com:Sub> gene segment with a human V<com:Sub>L< / com:Sub> gene segment, and an ectopic nucleotide sequence that encodes a mouse ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment is a <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment is a <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> gene segment.

[0087] In one embodiment, the mouse further comprises a human J<com:Sub>L< / com:Sub> gene segment, and the ectopic nucleotide sequence that encodes a mouse ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse is positioned between a human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment and the human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the mouse comprises a one or more human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segments and one or more human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segments and the ectopic nucleotide sequence that encodes a mouse ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse is positioned upstream (or 5') of the one or more human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segments. In a specific embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments are <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments.

[0088] In one embodiment, the mouse comprises an immunoglobulin heavy chain locus that comprises a deletion of an endogenous immunoglobulin locus nucleotide sequence that comprises an endogenous ADAM6 gene, comprises a nucleotide sequence encoding one or more human immunoglobulin gene segments, and wherein the ectopic nucleotide sequence encoding the mouse ADAM6 protein is within or directly adjacent to the nucleotide sequence encoding the one or more human immunoglobulin gene segments.

[0089] In one embodiment, the mouse comprises a replacement of all or substantially all endogenous V<com:Sub>H< / com:Sub> gene segments with a nucleotide sequence encoding one or more human V<com:Sub>L< / com:Sub> gene segments, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is within, or directly adjacent to, the nucleotide sequence encoding the one or more human V<com:Sub>L< / com:Sub> gene segments. In one embodiment, the mouse further comprises a replacement of one or more endogenous D<com:Sub>L< / com:Sub> gene segments with one or more human V<com:Sub>L< / com:Sub> and / or human J<com:Sub>L< / com:Sub> gene segments at the endogenous D<com:Sub>H< / com:Sub> gene locus. In one embodiment, the mouse further comprises a replacement of one or more endogenous J<com:Sub>H< / com:Sub> gene segments with one or more human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments at the endogenous <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> gene locus. In one embodiment, the mouse comprises a replacement of all or substantially all endogenous V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and J<com:Sub>H< / com:Sub> gene segments and a replacement at the endogenous V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and J<com:Sub>H< / com:Sub> gene loci with human V<com:Sub>L< / com:Sub> and J<com:Sub>L< / com:Sub> gene segments, wherein the mouse comprises an ectopic sequence encoding a mouse ADAM6 protein. In one embodiment, the mouse comprises an insertion of one or more human V<com:Sub>L< / com:Sub> and J<com:Sub>L< / com:Sub> gene segments at an endogenous immunoglobulin heavy chain locus, wherein the mouse comprises an ADAM6 gene that is functional in the mouse. In a specific embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments are <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In a specific embodiment, the ectopic sequence encoding the mouse ADAM6 protein is placed between the penultimate 3'-most V<com:Sub>L< / com:Sub> gene segment of the human V<com:Sub>L< / com:Sub> gene segments present, and the ultimate 5'-most <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segment of the human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments present. In a specific embodiment, the ectopic sequence encoding the mouse ADAM6 protein is placed upstream (or 5') of the 5'-most V<com:Sub>L< / com:Sub> gene segment of the human V<com:Sub>L< / com:Sub> gene segments present. In a specific embodiment, the mouse comprises a deletion of all or substantially all mouse V<com:Sub>H< / com:Sub> gene segments, and a replacement with at least 40 human V<com:Sub>L< / com:Sub> gene segments, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is placed downstream of human <com:Math><semantics>VK4−1<annotation encoding="application / x-tex">V_K4-1< / annotation>< / semantics>< / com:Math> gene segment and upstream of human <com:Math><semantics>JK1<annotation encoding="application / x-tex">J_K1< / annotation>< / semantics>< / com:Math> gene segment. In a specific embodiment, the mouse comprises a deletion of all or substantially all mouse V<com:Sub>H< / com:Sub> gene segments, and a replacement with at least 40 human V<com:Sub>L< / com:Sub> gene segments, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is placed upstream of a human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math>2-40 gene segment.

[0090] In a specific embodiment, the mouse comprises a replacement of all or substantially all endogenous V<com:Sub>H< / com:Sub> gene segments with a nucleotide sequence encoding one or more human V<com:Sub>L< / com:Sub> gene segments, and the ectopic nucleotide sequence encoding the mouse ADAM6 protein is within, or directly adjacent to, the nucleotide sequence encoding the one or more human V<com:Sub>L< / com:Sub> gene segments.

[0091] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segments are <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segments are <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> gene segments.

[0092] In one embodiment, the ectopic nucleotide sequence that encodes the mouse ADAM6 protein is present on a transgene in the genome of the mouse. In one embodiment, the ectopic nucleotide sequence that encodes the mouse ADAM6 protein is present extra- chromosomally in the mouse.

[0093] In one aspect, a mouse is provided that comprises a modification of an endogenous heavy chain immunoglobulin locus, wherein the mouse expresses a B cell that comprises a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence, and the B cell comprises in its genome (e.g., on a B cell chromosome) a gene encoding an ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human light chain V, J, and optionally a D gene sequence; a mouse heavy chain V, D, and / or J sequence; a human or mouse light chain V and / or J sequence. In one embodiment, the heavy chain constant region gene sequence comprises a human or a mouse heavy chain sequence selected from the group consisting of a C<com:Sub>H< / com:Sub>1, a hinge, a C<com:Sub>H< / com:Sub>2, a C<com:Sub>H< / com:Sub>3, and a combination thereof.

[0094] In one embodiment, the human light chain V and / or J sequence is selected from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J_\lambda< / annotation>< / semantics>< / com:Math> sequence.

[0095] In one aspect, a mouse is provided that comprises a functionally silenced endogenous immunoglobulin heavy chain locus, wherein ADAM6 function is maintained in the mouse, and further comprises an insertion of one or more human immunoglobulin gene segments, wherein the one or more human immunoglobulin gene segments include human V<com:Sub>L< / com:Sub> and JL gene segments, and optionally human D<com:Sub>H< / com:Sub> gene segments. In one embodiment, the one or more human immunoglobulin gene segments includes a human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_{\lambda}< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J_{\kappa}< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segments.

[0096] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a functionally silenced immunoglobulin light chain gene, and further comprises a replacement of one or more endogenous immunoglobulin heavy chain variable region gene segments with one or more human immunoglobulin light chain variable region gene segments, wherein the mouse lacks a functional endogenous ADAM6 locus, and wherein the mouse comprises an ectopic nucleotide sequence that expresses a mouse ADAM6 protein or an ortholog or homolog or fragment thereof that is functional in a male mouse.

[0097] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a functionally silenced immunoglobulin light chain gene locus, and further comprises a replacement of one or more endogenous immunoglobulin light chain variable gene segments with one or more human immunoglobulin light chain variable gene segments, wherein the mouse lacks a functional endogenous ADAM6 locus, and wherein the mouse comprises an ectopic nucleotide sequence that encodes a mouse ADAM6 protein or an ortholog or homolog or fragment thereof that is functional in a male mouse.

[0098] In one embodiment, the one or more human immunoglobulin light chain variable gene segments are contiguous with the ectopic nucleotide sequence.

[0099] In one aspect, a mouse is provided that lacks a functional endogenous ADAM6 locus or sequence and that comprises an ectopic nucleotide sequence encoding a mouse ADAM6 locus or functional fragment of a mouse ADAM6 locus or sequence, wherein the mouse is capable of mating with a mouse of the opposite sex to produce a progeny that comprises the ectopic ADAM6 locus or sequence. In one embodiment, the mouse is male. In one embodiment, the mouse is female.

[00100] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a human immunoglobulin light chain variable region gene segment at an endogenous immunoglobulin heavy chain variable region gene locus, the mouse lacks an endogenous functional ADAM6 sequence at the endogenous immunoglobulin heavy chain variable region gene locus, and wherein the mouse comprises an ectopic nucleotide sequence that expresses a mouse ADAM6 protein or an ortholog or homolog or fragment thereof that is functional in a male mouse.

[00101] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a human immunoglobulin light chain variable gene segment at an endogenous immunoglobulin heavy chain variable region gene locus, the mouse lacks an endogenous functional ADAM6 sequence at the endogenous immunoglobulin heavy chain variable gene locus, and wherein the mouse comprises an ectopic nucleotide sequence that expresses a mouse ADAM6 protein or an ortholog or homolog or fragment thereof that is functional in a male mouse.

[00102] In one embodiment, the ectopic nucleotide sequence that expresses the mouse ADAM6 protein is extra-chromosomal. In one embodiment, the ectopic nucleotide sequence that expresses the mouse ADAM6 protein is integrated at one or more loci in a genome of the mouse. In a specific embodiment, the one or more loci include an immunoglobulin locus.

[00103] In one aspect, a mouse is provided that expresses an immunoglobulin light chain sequence from a modified endogenous immunoglobulin heavy chain locus, wherein the heavy chain is derived from a human V<com:Sub>L< / com:Sub> gene segment, a J gene segment, and optionally a D<com:Sub>H< / com:Sub> gene segment, wherein the mouse comprises an ADAM6 activity that is functional in the mouse. In one embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment is selected from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> gene segment. In various embodiments, the J gene segment is a <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math>, a <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> or a <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segment or a combination thereof.

[00104] In one embodiment the mouse comprises a plurality of human V<com:Sub>L< / com:Sub> gene segments and a plurality of J gene segments. In a specific embodiment, the J gene segments are <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments.

[00105] In one aspect, a mouse is provided that expresses an immunoglobulin light chain sequence from a modified endogenous immunoglobulin heavy chain locus, wherein the heavy chain is derived from a human V<com:Sub>L< / com:Sub> gene segment and a J<com:Sub>L< / com:Sub> gene segment, wherein the mouse comprises an ADAM6 activity that is functional in the mouse.

[00106] In one embodiment, the mouse comprises a plurality of human V gene segments, a plurality of J gene segments, and optionally a plurality of D gene segments. In one embodiment, the D gene segments are human D gene segments. In one embodiment, the J gene segments are human J gene segments. In one embodiment, the mouse further comprises a humanized heavy chain constant region sequence, wherein the humanization comprises replacement of a sequence selected from a C<com:Sub>H< / com:Sub>1, hinge, C<com:Sub>H< / com:Sub>2, C<com:Sub>H< / com:Sub>3, and a combination thereof. In a specific embodiment, the heavy chain is derived from a human V gene segment, a human J gene segment, a human C<com:Sub>H< / com:Sub>1 sequence, a human or mouse hinge sequence, a mouse C<com:Sub>H< / com:Sub>2 sequence, and a mouse C<com:Sub>H< / com:Sub>3 sequence. In another specific embodiment, the mouse further comprises a human light chain constant sequence. In one embodiment, the mouse comprises an ADAM6 gene that is flanked 5' and 3' by endogenous immunoglobulin heavy chain gene segments. In a specific embodiment, the endogenous immunoglobulin heavy chain variable gene segments are incapable of encoding a heavy chain variable region of an antibody. In a specific embodiment, the ADAM6 gene of the mouse is at a position that is the same as in a wild-type mouse and the endogenous immunoglobulin heavy chain variable gene loci of the mouse are incapable of rearranging to encode a heavy chain of an antibody.

[00107] In one embodiment, the plurality of human V gene segments are flanked 5' (with respect to transcriptional direction of the V gene segments) by a sequence encoding an ADAM6 activity that is functional in the mouse. In a specific embodiment, the plurality of human V gene segments include human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments <com:Math><semantics>VK4−1<annotation encoding="application / x-tex">V_K4-1< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>VK5−2<annotation encoding="application / x-tex">V_K5-2< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>VK7−3<annotation encoding="application / x-tex">V_K7-3< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>VK2−4<annotation encoding="application / x-tex">V_K2-4< / annotation>< / semantics>< / com:Math>, Vκ1-5, Vκ1-6, Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15, Vκ1- 16, Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29, Vκ2-30, Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1- 37, <com:Math><semantics>VK2<annotation encoding="application / x-tex">V_K 2< / annotation>< / semantics>< / com:Math>-38, <com:Math><semantics>VK1<annotation encoding="application / x-tex">V_K 1< / annotation>< / semantics>< / com:Math>-39, and <com:Math><semantics>VK2<annotation encoding="application / x-tex">V_K 2< / annotation>< / semantics>< / com:Math>-40 and the human <com:Math><semantics>VK2<annotation encoding="application / x-tex">V_K 2< / annotation>< / semantics>< / com:Math>-40 gene segment is flanked 5' (with respect to transcriptional direction of the human Vκ2-40 gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse. In a specific embodiment, the sequence encoding an ADAM6 activity that is functional in the mouse is placed in the same transcription orientation with respect to the human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments. In a specific embodiment, the sequence encoding an ADAM6 activity that is functional in the mouse is placed in the opposite transcription orientation with respect to the human V<com:Sub>K< / com:Sub> gene segments.

[00108] In one embodiment, the V gene segment is flanked 3' (with respect to transcriptional direction of the V gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.

[00109] In one embodiment, the D gene segment is flanked 5' (with respect to transcriptional direction of the D gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.

[00110] In one embodiment, the J gene segment is flanked 5' (with respect to transcriptional direction of the J gene segment) by a sequence encoding an ADAM6 activity that is functional in the mouse.

[00111] In one embodiment, the ADAM6 activity that is functional in the mouse results from expression of a nucleotide sequence located 5' of the 5'-most D gene segment and 3' of the most 3'-most V gene segment (with respect to the direction of transcription of the V gene segment) of the modified endogenous heavy chain immunoglobulin locus.

[00112] In one embodiment, the ADAM6 activity that is functional in the mouse results from expression of a nucleotide sequence located 5' of the 5'-most J gene segment and 3' of the 3'-most V gene segment (with respect to the direction of transcription of the V gene segment) of the modified endogenous immunoglobulin heavy chain locus.

[00113] In one embodiment, the ADAM6 activity that is functional in the mouse results from expression of a nucleotide sequence located between two human V gene segments in the modified endogenous immunoglobulin heavy chain locus. In one embodiment, the two human V gene segments are a human <com:Math><semantics>Vκ5−2<annotation encoding="application / x-tex">V\kappa 5-2< / annotation>< / semantics>< / com:Math> gene segment and a <com:Math><semantics>Vκ4−1<annotation encoding="application / x-tex">V\kappa 4-1< / annotation>< / semantics>< / com:Math> gene segment.

[00114] In one embodiment, the ADAM6 activity that is functional in the mouse results from expression of a nucleotide sequence located between a human V gene segment and a human J gene segment in the modified endogenous immunoglobulin heavy chain locus. In one embodiment, the human V gene segment is a human <com:Math><semantics>Vκ4−1<annotation encoding="application / x-tex">V\kappa 4-1< / annotation>< / semantics>< / com:Math> gene segment and the human J segment is a Jκ1 gene segment.

[00115] In one embodiment, the nucleotide sequence comprises a sequence selected from a mouse ADAM6b sequence or functional fragment thereof, a mouse ADAM6a sequence or functional fragment thereof, and a combination thereof.

[00116] In various embodiments, the sequence encoding an ADAM6 activity that is functional in the mouse encodes a ADAM6b protein set forth in SEQ ID NO: 2 and / or encodes a ADAM6a protein set forth in SEQ ID NO: 1.

[00117] In one embodiment, the nucleotide sequence between the two human V gene segments is placed in opposite transcription orientation with respect to the human V gene segments. In a specific embodiment, nucleotide sequence encodes, from 5' to 3' with respect to the direction of transcription of ADAM6 genes, an ADAM6a sequence followed by an ADAM6b sequence. In one embodiment, t

[00118] In one embodiment, the nucleotide sequence between the human V gene segment and the human J gene segment is placed in opposite transcription orientation with respect to the human V and J gene segments. In a specific embodiment, the nucleotide sequence encodes, from 5' to 3' with respect to the direction of transcription of ADAM6 genes, an ADAM6a sequence followed by an ADAM6b sequence.

[00119] In one embodiment, the mouse comprises a hybrid immunoglobulin sequence, wherein the hybrid immunoglobulin sequence comprises a human immunoglobulin κ light chain sequence contiguous with a non-human ADAM6 sequence.

[00120] In one embodiment, the mouse comprises a human sequence contiguous with a mouse sequence at an endogenous immunoglobulin heavy chain locus, wherein the contiguous sequence comprises at least one human V<com:Sub>L< / com:Sub> gene segment, a mouse ADAM6 sequence or ortholog or homolog or functional fragment thereof, and a human J<com:Sub>L< / com:Sub> gene segment. In a specific embodiment, the mouse ADAM6 sequence or ortholog or homolog or functional fragment thereof is positioned immediately adjacent to the at least one human V<com:Sub>L< / com:Sub> gene segment. In one embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the mouse ADAM6 sequence or ortholog or homolog or functional fragment thereof is positioned immediately adjacent and 3' to the at least one human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment and immediately adjacent and 5' to the human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segment. In a specific embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment and the human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segment is a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment.

[00121] In one embodiment, the sequence encoding the ADAM6 activity that is functional in the mouse is a mouse ADAM6 sequence or functional fragment thereof.

[00122] In one embodiment, the mouse comprises an endogenous mouse DFL16.1 gene segment (e.g., in a mouse heterozygous for the modified endogenous mouse immunoglobulin heavy chain locus), or a human D<com:Sub>H< / com:Sub>1-1 gene segment. In one embodiment, the D gene segment of the immunoglobulin heavy chain expressed by the mouse is derived from an endogenous mouse DFL16.1 gene segment or a human D<com:Sub>H< / com:Sub>1-1 gene segment.

[00123] In one aspect, a mouse is provided that comprises a nucleic acid sequence encoding a mouse ADAM6 (or homolog or ortholog or functional fragment thereof) in a DNA- bearing cell of non-rearranged B cell lineage, but does not comprise the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) in a B cell that comprise rearranged immunoglobulin loci, wherein the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) occurs in the genome at a position that is different from a position in which a mouse ADAM6 gene appears in a wild-type mouse. In one embodiment, the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is present in all or substantially all DNA-bearing cells that are not of rearranged B cell lineage; in one embodiment, the nucleic acid sequence is present in germline cells of the mouse, but not in a chromosome of a rearranged B cell.

[00124] In one aspect, a mouse is provided that comprises a nucleic acid sequence encoding a mouse ADAM6 (or homolog or ortholog or functional fragment thereof) in all or substantially all DNA-bearing cells, including B cells that comprise rearranged immunoglobulin loci, wherein the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) occurs in the genome at a position that is different from a position in which a mouse ADAM6 gene appears in a wild-type mouse. In one embodiment, the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is on a nucleic acid that is contiguous with the rearranged immunoglobulin locus. In one embodiment, the nucleic acid that is contiguous with the rearranged immunoglobulin locus is a chromosome. In one embodiment, the chromosome is a chromosome that is found in a wild-type mouse and the chromosome comprises a modification of a mouse immunoglobulin locus.

[00125] In one aspect, a genetically modified mouse is provided, wherein the mouse comprises a B cell that comprises in its genome an ADAM6 sequence or ortholog or homolog thereof. In one embodiment, the ADAM6 sequence or ortholog or homolog thereof is at an immunoglobulin heavy chain locus. In a specific embodiment, the heavy chain locus comprises endogenous immunoglobulin heavy chain gene segments that are incapable of rearranging to encode the heavy chain of an antibody. In one embodiment, the ADAM6 sequence or ortholog or homolog thereof is at a locus that is not an immunoglobulin locus. In one embodiment, the ADAM6 sequence is on a transgene driven by a heterologous promoter. In a specific embodiment, the heterologous promoter is a non-immunoglobulin promoter. In a specific embodiment, B cell expresses an ADAM6 protein or ortholog or homolog thereof.

[00126] In one embodiment, 90% or more of the B cells of the mouse comprise a gene encoding an ADAM6 protein or an ortholog thereof or a homolog thereof or a fragment thereof that is functional in the mouse. In a specific embodiment, the mouse is a male mouse.

[00127] In one embodiment, the B cell genome comprises a first allele and a second allele comprising the ADAM6 sequence or ortholog or homolog thereof. In one embodiment, the B cell genome comprises a first allele but not a second allele comprising the ADAM6 sequence or ortholog or homolog thereof.

[00128] In one aspect, a mouse is provided that comprises a modification at one or more endogenous immunoglobulin heavy chain alleles, wherein the modification maintains one or more endogenous ADAM6 alleles.

[00129] In one embodiment, the modification renders the mouse incapable of expressing a functional heavy chain that comprises rearranged endogenous heavy chain gene segments from at least one heavy chain allele and maintains an endogenous ADAM6 allele located within the at least one endogenous immunoglobulin heavy chain allele.

[00130] In one embodiment, the mice are incapable of expressing a functional heavy chain that comprises rearranged endogenous heavy chain gene segments from at least one of the endogenous immunoglobulin heavy chain alleles, and the mice express and ADAM6 protein from an endogenous ADAM6 allele. In a specific embodiment, the mice are incapable of expressing a functional heavy chain that comprises rearranged endogenous heavy chain gene segments from two endogenous immunoglobulin heavy chain alleles, and the mice express an ADAM6 protein from one or more endogenous ADAM6 alleles.

[00131] In one embodiment, the mice are incapable of expressing a functional heavy chain from each endogenous heavy chain allele, and the mice comprise a functional ADAM6 allele located within 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 or more Mbp upstream (with respect to the direction of transcription of the mouse immunoglobulin heavy chain locus) of a mouse immunoglobulin heavy chain constant region sequence. In a specific embodiment, the functional ADAM6 allele is at the endogenous heavy chain locus (e.g., in an intergenic V-D region, between two V gene segments, between a V and a D gene segment, between a D and a J gene segment, etc.). In a specific embodiment, the functional ADAM6 allele is located within a 90 to 100 kb intergenic sequence between the final mouse V gene segment and the first mouse D gene segment. In another specific embodiment, the endogenous 90 to 100 kb intergenic V-D sequence is removed, and the ectopic ADAM6 sequence is placed between the final V and the first D gene segment.

[00132] In one aspect, a mouse is provided that comprises a modification at one or more endogenous ADAM6 alleles.

[00133] In one embodiment, the modification renders the mouse incapable of expressing a functional ADAM6 protein from at least one of the one or more endogenous ADAM6 alleles. In a specific embodiment, the mouse is incapable of expressing a functional ADAM6 protein from each of the endogenous ADAM6 alleles.

[00134] In one embodiment, the mouse is incapable of expressing a functional ADAM6 protein from each endogenous ADAM6 allele, and the mice comprise an ectopic ADAM6 sequence.

[00135] In one embodiment, the mouse is incapable of expressing a functional ADAM6 protein from each endogenous ADAM6 allele, and the mouse comprises an ectopic ADAM6 sequence located within 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 or more kb upstream (with respect to the direction of transcription of the mouse heavy chain locus) of a mouse immunoglobulin heavy chain constant region sequence. In a specific embodiment, the ectopic ADAM6 sequence is at the endogenous immunoglobulin heavy chain locus (e.g., in an intergenic V-D region, between two V gene segments, between a V and a J gene segment, between a D and a J gene segment, etc.). In a specific embodiment, the ectopic ADAM6 sequence is located within a 90 to 100 kb intergenic sequence between the final mouse V gene segment and the first mouse D gene segment. In another specific embodiment, the endogenous 90 to 100 kb intergenic V-D sequence is removed, and the ectopic ADAM6 sequence is placed between a human Vκ gene segment and a first human Jk gene segment. In another specific embodiment, the endogenous 90 to 100 kb intergenic V-D sequence is removed, and the ectopic ADAM6 sequence is placed 5' or upstream of a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment, wherein the human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment is selected from a human <com:Math><semantics>VK4−1<annotation encoding="application / x-tex">V_K4-1< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>VK2−40<annotation encoding="application / x-tex">V_K2-40< / annotation>< / semantics>< / com:Math> gene segment.

[00136] In one embodiment, the mouse is capable of expressing a functional ADAM6 protein from one or more endogenous ADAM6 alleles and the modification includes an insertion of a human sequence that encodes an immunoglobulin variable domain. In one embodiment, the human sequence comprises unrearranged immunoglobulin gene segments. In a specific embodiment, the human sequence comprises a V gene segment and a J gene segment. In another specific embodiment, the human sequence comprises a V gene segment, a J gene segment, and a D gene segment.

[00137] In one aspect, an infertile male mouse is provided, wherein the mouse comprises a deletion of two or more endogenous ADAM6 alleles. In one aspect, a female mouse is provided that is a carrier of a male infertility trait, wherein the female mouse comprises in its germline a nonfunctional ADAM6 allele or a knockout of an endogenous ADAM6 allele.

[00138] In one aspect, a mouse comprising an endogenous immunoglobulin heavy chain V, D, and or J gene segment that are incapable of rearranging to encode an heavy chain of an antibody is provided, wherein the majority of the B cells of the mouse comprise an functional ADAM6 gene. In various embodiments, the majority of the B cells further comprise one or more human V<com:Sub>L< / com:Sub> and J<com:Sub>L< / com:Sub> gene segments upstream of a mouse immunoglobulin heavy chain constant region. In one embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments are <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments.

[00139] In one embodiment, the mouse comprises an intact endogenous immunoglobulin heavy chain V, D, and J gene segments that are incapable of rearranging to encode a functional heavy chain of an antibody. In one embodiment, the mouse comprises at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments, and a combination thereof; wherein the at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments are incapable of rearranging to encode a heavy chain variable region of an antibody. In a specific embodiment, the mouse comprises a functional ADAM6 gene located within the intact endogenous immunoglobulin heavy chain V, D, and J gene segments. In one embodiment, the mouse comprises an endogenous heavy chain locus that includes an endogenous ADAM6 locus, wherein the endogenous heavy chain locus comprises 89 V gene segments, 13 D gene segments, and four J gene segments, wherein the endogenous heavy chain gene segments are incapable of rearranging to encode a heavy chain variable region of an antibody and the ADAM6 locus encodes an ADAM6 protein that is functional in the mouse.

[00140] In one aspect, a method is provided for making an infertile male mouse, comprising rendering an endogenous ADAM6 allele of a donor ES cell nonfunctional (or knocking out said allele), introducing the donor ES cell into a host embryo, gestating the host embryo in a surrogate mother, and allowing the surrogate mother to give birth to progeny derived in whole or in part from the donor ES cell. In one embodiment, the method further comprises breeding progeny to obtain an infertile male mouse.

[00141] In one aspect, a method is provided for making a mouse with a genetic modification of interest, wherein the mouse is infertile, the method comprising the steps of (a) making a genetic modification of interest in a genome; (b) modifying the genome to knockout an endogenous ADAM6 allele, or render an endogenous ADAM6 allele nonfunctional; and, (c) employing the genome in making a mouse. In various embodiments, the genome is from an ES cell or used in a nuclear transfer experiment.

[00142] In one aspect, a mouse that lacks an endogenous immunoglobulin heavy chain V, D, and J gene segment is provided, wherein a majority of the B cells of the mouse comprise an ADAM6 sequence or ortholog or homolog thereof.

[00143] In one embodiment, the mouse lacks endogenous immunoglobulin heavy chain gene segments selected from two or more V gene segments, two or more D gene segments, two or more J gene segments, and a combination thereof. In one embodiment, the mouse lacks immunoglobulin heavy chain gene segments selected from at least one and up to 89 V gene segments, at least one and up to 13 D gene segments, at least one and up to four J gene segments, and a combination thereof. In one embodiment, the mouse lacks a genomic DNA fragment from chromosome 12 comprising about three megabases of the endogenous immunoglobulin heavy chain locus. In a specific embodiment, the mouse lacks all functional endogenous heavy chain V, D, and J gene segments. In a specific embodiment, the mouse lacks 89 V<com:Sub>H< / com:Sub> gene segments, 13 D<com:Sub>H< / com:Sub> gene segments and four J<com:Sub>H< / com:Sub> gene segments.

[00144] In one aspect, a mouse is provided, wherein the mouse has a genome in the germline comprising a modification of an immunoglobulin heavy chain locus, wherein the modification to the immunoglobulin heavy chain locus comprises the replacement of one or more mouse immunoglobulin variable region sequences with one or more non-mouse immunoglobulin variable region sequences, and wherein the mouse comprises a nucleic acid sequence encoding a mouse ADAM6 protein. In one embodiment, the D<com:Sub>H< / com:Sub> and J<com:Sub>H< / com:Sub> sequences and at least 3, at least 10, at least 20, at least 40, at least 60, or at least 80 V<com:Sub>H< / com:Sub> sequences of the endogenous immunoglobulin heavy chain locus are replaced by non- mouse immunoglobulin light chain sequences. In one embodiment, the D<com:Sub>H< / com:Sub>, J<com:Sub>H< / com:Sub>, and all V<com:Sub>H< / com:Sub> sequences of the endogenous immunoglobulin heavy chain locus are replaced by a plurality of non-mouse immunoglobulin V<com:Sub>L< / com:Sub> gene segments, one or more J<com:Sub>L< / com:Sub> gene segment, and optionally one or more D gene segment sequences. The non-mouse immunoglobulin sequences can be unrearranged. In one embodiment, the non-mouse immunoglobulin sequences comprise complete unrearranged <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> regions of the non-mouse species. In one embodiment, the non-mouse immunoglobulin sequences are capable of forming a complete variable region, i.e., a rearranged variable region containing V<com:Sub>L< / com:Sub> and J<com:Sub>L< / com:Sub> gene segments joined together to form an sequence that encodes a light chain variable region, of the non-mouse species, operably linked to one or more endogenous constant regions. The non-mouse species can be Homo sapiens and the non-mouse immunoglobulin sequences can be human sequences.

[00145] In one aspect, a genetically modified mouse comprising a nucleotide sequence encoding an ADAM6 protein or functional fragment thereof that is contiguous with a human immunoglobulin light chain variable gene segment is provided.

[00146] In one embodiment, the mouse lacks an endogenous unmodified ADAM6 gene sequence. In one embodiment, the mouse lacks a functional endogenous ADAM6 gene sequence.

[00147] In one embodiment, the human immunoglobulin light chain variable gene segment is an immunoglobulin <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable gene segment. In one embodiment, the human immunoglobulin light chain variable gene segment is an immunoglobulin <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable gene segment. In one embodiment, the human immunoglobulin light chain variable gene segment is operably linked to an immunoglobulin heavy chain constant gene sequence.

[00148] In one embodiment, the immunoglobulin heavy chain constant gene sequence is a mouse or rat or human heavy chain gene sequence. In one embodiment, the heavy chain constant gene sequence comprises a C<com:Sub>H< / com:Sub>1 and / or a hinge region.

[00149] In one embodiment, the mouse comprises deletion of, or replacement of, one or more endogenous immunoglobulin heavy chain gene sequences.

[00150] In one embodiment, the mouse further comprises an unrearranged human Vκ or unrearranged human Vλ gene segment operably linked to a human or mouse or rat light chain constant region sequence. In one embodiment, the mouse comprises a plurality of unrearranged human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments (e.g., two or more human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segments and one or more human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segments) or a plurality of unrearranged human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_{\lambda}< / annotation>< / semantics>< / com:Math> gene segments (e.g., two or more human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segments and one or more human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> segments). In one embodiment, the unrearranged human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or unrearranged human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> gene segments are operably linked to a constant region sequence at an endogenous immunoglobulin light chain locus.

[00151] In one embodiment, the mouse further comprises a modification that renders an endogenous <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain locus and / or an endogenous <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus nonfunctional. In one embodiment, the mouse comprises a knockout or a deletion of an endogenous mouse <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> and / or an endogenous mouse <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus.

[00152] In one aspect, a method for maintaining a mouse strain is provided, wherein the mouse strain comprises a replacement of a mouse immunoglobulin heavy chain sequence with one or more human immunoglobulin light chain sequences. In one embodiment, the one or more human immunoglobulin light chain sequences are human immunoglobulin <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments.

[00153] In one embodiment, the mouse strain comprises a deletion of one or more mouse V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and / or J<com:Sub>H< / com:Sub> gene segments. In one embodiment, the mouse further comprises one or more human V<com:Sub>L< / com:Sub> gene segments and one or more human J<com:Sub>L< / com:Sub> gene segments. In one embodiment, the mouse comprises at least 6, at least 16, at least 30, or at least 40 human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> gene segments and at least five <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J_{\kappa}< / annotation>< / semantics>< / com:Math> gene segments. In a specific embodiment, the human light chain gene segments are operably linked to a constant region gene. In one embodiment, the constant region gene is a mouse constant region gene. In one embodiment, the constant region gene comprises a mouse constant region gene sequence selected from a C<com:Sub>H< / com:Sub>1, a hinge, a C<com:Sub>H< / com:Sub>2, a C<com:Sub>H< / com:Sub>3, and / or a C<com:Sub>H< / com:Sub>4 or a combination thereof.

[00154] In one embodiment, the method comprises generating a male mouse heterozygous for the replacement of the mouse immunoglobulin heavy chain sequence, and breeding the heterozygous male mouse with a wild-type female mouse or a female mouse that is homozygous or heterozygous for the human heavy chain sequence. In one embodiment, the method comprises maintaining the strain by repeatedly breeding heterozygous males with females that are wild type or homozygous or heterozygous for the human heavy chain sequence.

[00155] In one embodiment, the method comprises obtaining cells from male or female mice homozygous or heterozygous for the human heavy chain sequence, and employing those cells as donor cells or nuclei therefrom as donor nuclei, and using the cells or nuclei to make genetically modified animals using host cells and / or gestating the cells and / or nuclei in surrogate mothers.

[00156] In one embodiment, only male mice that are heterozygous for the replacement at the heavy chain locus are bred to female mice. In a specific embodiment, the female mice are homozygous, heterozygous, or wild type with respect to a replaced heavy chain locus.

[00157] In one embodiment, the mouse further comprises a replacement of <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable sequences at an endogenous immunoglobulin light chain locus with heterologous immunoglobulin light chain sequences. In one embodiment, the heterologous immunoglobulin light chain sequences are human immunoglobulin <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable sequences.

[00158] In one embodiment, the mouse further comprises a transgene at a locus other than an endogenous immunoglobulin locus, wherein the transgene comprises a sequence encoding a rearranged or unrearranged heterologous <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain sequence (e.g., unrearranged V<com:Sub>L< / com:Sub> and unrearranged J<com:Sub>L< / com:Sub>, or rearranged VJ) operably linked (for unrearranged) or fused (for rearranged) to an immunoglobulin light chain constant region sequence. In one embodiment, the heterologous <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain sequence is human. In one embodiment, the constant region sequence is selected from rodent, human, and non-human primate. In one embodiment, the constant region sequence is selected from mouse, rat, and hamster. In one embodiment, the transgene comprises a non-immunoglobulin promoter that drives expression of the light chain sequences. In a specific embodiment, the promoter is a transcriptionally active promoter. In a specific embodiment, the promoter is a ROSA26 promoter.

[00159] In one aspect, a fertile mouse is provided that comprises a modification of an endogenous ADAM6 gene, wherein the mouse comprises an ectopic sequence that confers ADAM6 function to the mouse, and wherein the mouse comprises in its germline an unrearranged human immunoglobulin light chain gene segment operably linked to a nucleic acid sequence encoding an immunoglobulin heavy chain sequence.

[00160] In one aspect, a fertile mouse is provided that comprises a modification of an endogenous ADAM6 locus, wherein the modification renders the ADAM6 locus nonfunctional, and wherein the mouse expresses an immunoglobulin binding protein comprising a human immunoglobulin light chain variable domain contiguous with a heavy chain constant sequence.

[00161] In one embodiment, the immunoglobulin binding protein further comprises a cognate human immunoglobulin light chain variable domain fused with a light chain constant sequence.

[00162] In one embodiment, the heavy chain constant sequence and the light chain constant sequence are non-human.

[00163] In one aspect, a mouse is provided, comprising an immunoglobulin heavy chain locus comprising a replacement of one or more immunoglobulin heavy chain variable region <com:Math><semantics>(VH)<annotation encoding="application / x-tex">(V_H)< / annotation>< / semantics>< / com:Math> gene segments, heavy chain diversity <com:Math><semantics>(DH)<annotation encoding="application / x-tex">(D_H)< / annotation>< / semantics>< / com:Math> gene segments, and heavy chain joining <com:Math><semantics>(JH)<annotation encoding="application / x-tex">(J_H)< / annotation>< / semantics>< / com:Math> gene segments at an endogenous immunoglobulin heavy chain locus with one or more light chain variable region <com:Math><semantics>(VL)<annotation encoding="application / x-tex">(V_L)< / annotation>< / semantics>< / com:Math> gene segments and one or more light chain joining region <com:Math><semantics>(JL)<annotation encoding="application / x-tex">(J_L)< / annotation>< / semantics>< / com:Math> gene segments, wherein the mouse is capable of expressing an ADAM6 protein.

[00164] In one aspect, a mouse is provided, comprising an immunoglobulin heavy chain locus that comprises a replacement of all or substantially all V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and J<com:Sub>H< / com:Sub> gene segments with one or more V<com:Sub>L< / com:Sub> gene segments and one or more J<com:Sub>L< / com:Sub> gene segments to form a V<com:Sub>I< / com:Sub> gene segment sequence at an endogenous heavy chain locus (VL<com:Sub>H< / com:Sub> locus), wherein the VL<com:Sub>H< / com:Sub> locus is capable of recombining with an endogenous C<com:Sub>H< / com:Sub> gene to form a rearranged gene that is derived from a V<com:Sub>L< / com:Sub> gene segment, a J<com:Sub>L< / com:Sub> gene segment, and an endogenous C<com:Sub>H< / com:Sub> gene.

[00165] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math>. In one embodiment, the <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math>. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> and human J<com:Sub>L< / com:Sub> segments.

[00166] In one embodiment, all or substantially all <com:Math><semantics>VH<annotation encoding="application / x-tex">V_H< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> gene segments are replaced with at least six human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments and at least one <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, all or substantially all <com:Math><semantics>VH<annotation encoding="application / x-tex">V_H< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> gene segments are replaced with at least 16 human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> gene segments (human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math>) and at least one <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, all or substantially all <com:Math><semantics>VH<annotation encoding="application / x-tex">V_H< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math>, and <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> gene segments are replaced with at least 30 human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments and at least one <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, all or substantially all V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and J<com:Sub>H< / com:Sub> gene segments are replaced with at least 40 human V<com:Sub>K< / com:Sub> gene segments and at least one <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the at least one <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segment comprises two, three, four, or five human <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segments.

[00167] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segments. In one embodiment, the human V<com:Sub>K< / com:Sub> segments comprise 4-1, 5-2, 7-3, 2-4, 1-5, and 1-6. In one embodiment, the human V<com:Sub>K< / com:Sub> segments comprise 3-7, 1-8, 1-9, 2-10, 3-11, 1-12, 1-13, 2-14, 3-15, and 1-16. In one embodiment, the human V<com:Sub>κ< / com:Sub> segments comprise 1-17, 2-18, 2-19, 3- 20, 6-21, 1-22, 1-23, 2-24, 3-25, 2-26, 1-27, 2-28, 2-29, and 2-30. In one embodiment, the human V<com:Sub>κ< / com:Sub> segments comprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2- 40.

[00168] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segments and comprise 4-1, 5-2, 7-3, 2-4, 1-5, 1-6, 3-7, 1-8, 1-9, 2-10, 3-11, 1-12, 1-13, 2-14, 3-15, and 1-16. In one embodiment, the Vκ segments further comprise 1-17, 2-18, 2-19, 3-20, 6-21, 1-22, 1-23, 2- 24, 3-25, 2-26, 1-27, 2-28, 2-29, and 2-30. In one embodiment, the Vκ segments further comprise 3-31, 1-32, 1-33, 3-34, 1-35, 2-36, 1-37, 2-38, 1-39, and 2-40.

[00169] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segments and comprise a fragment of cluster A of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus. In a specific embodiment, the fragment of cluster A of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus extends from hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>3-27 through hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>3- 1.

[00170] In one embodiment, the V<com:Sub>L< / com:Sub> segments comprise a fragment of cluster B of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus. In a specific embodiment, the fragment of cluster B of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus extends from hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>5-52 through hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>1-40.

[00171] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments comprise a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence that comprises a genomic fragment of cluster A and a genomic fragment of cluster B. In a one embodiment, the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence comprises at least one gene segment of cluster A and at least one gene segment of cluster B.

[00172] In one embodiment, the V<com:Sub>L< / com:Sub> segments comprise at least one gene segment of cluster B and at least one gene segment of cluster C.

[00173] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments comprise hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments comprise a contiguous sequence of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus that spans from <com:Math><semantics>Vλ3<annotation encoding="application / x-tex">V\lambda 3< / annotation>< / semantics>< / com:Math>-12 to <com:Math><semantics>Vλ3<annotation encoding="application / x-tex">V\lambda 3< / annotation>< / semantics>< / com:Math>-1. In one embodiment, the contiguous sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hVλs. In a specific embodiment, the hVλs include 3-1, 4-3, 2-8, 3-9, 3-10, 2-11, and 3-12. In a specific embodiment, the hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>s comprises a contiguous sequence of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> locus that spans from <com:Math><semantics>Vλ3−12<annotation encoding="application / x-tex">V\lambda 3-12< / annotation>< / semantics>< / com:Math> to <com:Math><semantics>Vλ3−1<annotation encoding="application / x-tex">V\lambda 3-1< / annotation>< / semantics>< / com:Math>.

[00174] In one embodiment, the hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>s comprises 13 to 28 or more hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>s. In a specific embodiment, the hVλs include 2-14, 3-16, 2-18, 3-19, 3-21, 3-22, 2-23, 3-25, and 3-27. In a specific embodiment, the hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>s comprise a contiguous sequence of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> locus that spans from <com:Math><semantics>Vλ3−27<annotation encoding="application / x-tex">V\lambda 3-27< / annotation>< / semantics>< / com:Math> to <com:Math><semantics>Vλ3−1<annotation encoding="application / x-tex">V\lambda 3-1< / annotation>< / semantics>< / com:Math>.

[00175] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments comprise 29 to 40 hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>s. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments comprise a contiguous sequence of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> locus that spans from <com:Math><semantics>Vλ3<annotation encoding="application / x-tex">V\lambda 3< / annotation>< / semantics>< / com:Math>-29 to <com:Math><semantics>Vλ3<annotation encoding="application / x-tex">V\lambda 3< / annotation>< / semantics>< / com:Math>-1, and a contiguous sequence of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> locus that spans from <com:Math><semantics>Vλ5<annotation encoding="application / x-tex">V\lambda 5< / annotation>< / semantics>< / com:Math>-52 to <com:Math><semantics>Vλ1<annotation encoding="application / x-tex">V\lambda 1< / annotation>< / semantics>< / com:Math>-40. In a specific embodiment, all or substantially all sequence between hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>1-40 and hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>3-29 in the genetically modified mouse consists essentially of a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> sequence of approximately 959 bp found in nature (e.g., in the human population) downstream of the hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>1-40 gene segment (downstream of the 3' untranslated portion), a restriction enzyme site (e.g., Pl-Scel), followed by a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> sequence of approximately 3,431 bp upstream of the hV<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>3-29 gene segment found in nature.

[00176] In one embodiment, the J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> is human and is selected from the group consisting of Jκ1, Jκ2, Jκ3, Jκ4, Jκ5, and a combination thereof. In a specific embodiment, the Jκ comprises Jκ1 through Jκ5.

[00177] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segments, and the <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segment comprises an RSS having a 12-mer spacer, wherein the RSS is juxtaposed at the upstream end of the J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segments are human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> and the <com:Math><semantics>VLH<annotation encoding="application / x-tex">VL_H< / annotation>< / semantics>< / com:Math> locus comprises two or more <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segments, each comprising an RSS having a 12-mer spacer wherein the RSS is juxtaposed at the upstream end of each J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segment.

[00178] In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments comprise contiguous human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segments spanning the human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> locus from V<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>4-1 through V<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>2-40, and the J<com:Sub>L< / com:Sub> segments comprise contiguous gene segments spanning the human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> locus from J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>1 through J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>5.

[00179] In one embodiment, where the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segments are <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segments and no <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> segment is present between the V<com:Sub>L< / com:Sub> segments and J segments, the V<com:Sub>L< / com:Sub> segments are flanked downstream (i.e., juxtaposed on the downstream side) with 23-mer RSS, and J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> segments if present or <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> segments if present are flanked upstream (i.e., juxtaposed on the upstream side) with 12-mer RSS.

[00180] In one embodiment, where the V gene segments are <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments and no <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> gene segment is present between the V gene segments and J gene segments, the <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments are each juxtaposed on the downstream side with a 12-mer RSS, and J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> segments if present or J<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> segments if present are each juxtaposed on the upstream side with a 23-mer RSS.

[00181] In one embodiment, the mouse comprises a rearranged gene that is derived from a V<com:Sub>L< / com:Sub> gene segment, a J<com:Sub>L< / com:Sub> gene segment, and an endogenous C<com:Sub>H< / com:Sub> gene. In one embodiment, the rearranged gene is somatically mutated. In one embodiment, the rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions. In one embodiment, the N additions and / or the somatic mutations observed in the rearranged gene derived from the V<com:Sub>L< / com:Sub> segment and the J<com:Sub>L< / com:Sub> segment are 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or at least 5-fold more than the number of N additions and / or somatic mutations observed in a rearranged light chain variable domain (derived from the same V<com:Sub>L< / com:Sub> gene segment and the same J<com:Sub>L< / com:Sub> gene segment) that is rearranged at an endogenous light chain locus. In one embodiment, the rearranged gene is in a B cell that specifically binds an antigen of interest, wherein the B cell binds the antigen of interest with a K<com:Sub>D< / com:Sub> in the low nanomolar range or lower <com:Math><semantics>(e.g.,aKD)<annotation encoding="application / x-tex">(e.g., a K_D)< / annotation>< / semantics>< / com:Math> of 10 nanomolar or lower). In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment, the <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segment, or both, are human gene segments. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segments are human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the mouse <com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math> gene is selected from IgM, IgD, IgG, IgA and IgE. In a specific embodiment, the mouse IgG is selected from IgG1, IgG2A, IgG2B, IgG2C and IgG3. In another specific embodiment, the mouse IgG is lgG1.

[00182] In one embodiment, the mouse comprises a B cell, wherein the B cell expresses from a locus on a chromosome of the B cell a binding protein consisting essentially of four polypeptide chains, wherein the four polypeptide chains consist essentially of (a) two identical polypeptides that comprise an endogenous C<com:Sub>H< / com:Sub> region fused with a V<com:Sub>L< / com:Sub>; and, (b) two identical polypeptides that comprise an endogenous C<com:Sub>L< / com:Sub> region fused with a V<com:Sub>L< / com:Sub> region that is cognate with respect to the V<com:Sub>L< / com:Sub> region that is fused with the mouse C<com:Sub>H< / com:Sub> region, and, in one embodiment, is a human (e.g., a human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>) <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> region. In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> region fused to the endogenous C<com:Sub>H< / com:Sub> region is a human V<com:Sub>L< / com:Sub> region. In a specific embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> region fused with the mouse <com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math> region is a <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> region. In a specific embodiment, the human V<com:Sub>L< / com:Sub> region fused with the mouse C<com:Sub>H< / com:Sub> region is identical to a V region encoded by a rearranged human germline light chain nucleotide sequence. In a specific embodiment, the human V<com:Sub>L< / com:Sub> region fused to the mouse C<com:Sub>H< / com:Sub> region comprises two, three, four, five, six, or more somatic hypermutations. In one embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> region fused to the mouse <com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math> region is encoded by a rearranged gene that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00183] In one embodiment, at least 50% of all IgG molecules expressed by the mouse comprise a polypeptide that comprises an IgG isotype C<com:Sub>H< / com:Sub> region and a V<com:Sub>L< / com:Sub> region, wherein the length of said polypeptide is no longer than 535, 530, 525, 520, or 515 amino acids. In one embodiment, at least 75% of all IgG molecules comprise the polypeptide recited in this paragraph. In one embodiment, at least 80%, 85%, 90%, or 95% of all IgG molecules comprise the polypeptide recited in this paragraph. In a specific embodiment, all IgG molecules expressed by the mouse comprise a polypeptide that is no longer than the length of the polypeptide recited in this paragraph.

[00184] In one embodiment, the mouse expresses a binding protein comprising a first polypeptide that comprises an endogenous C<com:Sub>H< / com:Sub> region fused with a variable domain encoded by a rearranged human V gene segment and a J gene segment but not a D<com:Sub>H< / com:Sub> gene segment, and a second polypeptide that comprises an endogenous C<com:Sub>L< / com:Sub> region fused with a V domain encoded by a rearranged human V gene segment and a J gene segment but not a D<com:Sub>H< / com:Sub> gene segment, and the binding protein specifically binds an antigen with an affinity in the micromolar, nanomolar, or picomolar range. In one embodiment, the J segment is a human J segment (e.g., a human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segment). In one embodiment, the human V segment is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment. In one embodiment, the variable domain that is fused with the endogenous C<com:Sub>H< / com:Sub> region comprises a greater number of somatic hypermutations than the variable region that is fused with the endogenous C<com:Sub>L< / com:Sub> region; in a specific embodiment, the variable region fused with the endogenous C<com:Sub>H< / com:Sub> region comprises about 1.5, 2-, 3-, 4-, or 5- fold or more somatic hypermutations than the V region fused to the endogenous C<com:Sub>L< / com:Sub> region; in a specific embodiment, the V region fused with the mouse C<com:Sub>H< / com:Sub> region comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more somatic hypermutations than the V region fused with the mouse C<com:Sub>L< / com:Sub> region. In one embodiment, the V region fused with the mouse C<com:Sub>H< / com:Sub> region is encoded by a rearranged gene that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00185] In one embodiment, the mouse expresses a binding protein comprising a first light chain variable domain (V<com:Sub>L< / com:Sub>1) fused with an immunoglobulin heavy chain constant region sequence and a second light chain variable domain (V<com:Sub>L< / com:Sub>2) fused with an immunoglobulin light chain constant region, wherein V<com:Sub>L< / com:Sub>1 comprises a number of somatic hypermutations that is about 1.5- to about 5-fold higher or more than the number of somatic hypermutations present in <com:Math><semantics>VL2<annotation encoding="application / x-tex">V_L 2< / annotation>< / semantics>< / com:Math>. In one embodiment, the number of somatic hypermutations in <com:Math><semantics>VL1<annotation encoding="application / x-tex">V_L 1< / annotation>< / semantics>< / com:Math> is about 2- to about 4-fold higher than in <com:Math><semantics>VL2<annotation encoding="application / x-tex">V_L2< / annotation>< / semantics>< / com:Math>. In one embodiment, the number of somatic hypermutations in <com:Math><semantics>VL1<annotation encoding="application / x-tex">V_L 1< / annotation>< / semantics>< / com:Math> is about 2- to about 3-fold higher than in <com:Math><semantics>VL2<annotation encoding="application / x-tex">V_L 2< / annotation>< / semantics>< / com:Math>. In one embodiment, V<com:Sub>L< / com:Sub>1 is encoded by a sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00186] In one aspect, a genetically modified mouse is provided that expresses an immunoglobulin that consists essentially of the following polypeptides: a first two identical polypeptides that each consists essentially of a C<com:Sub>H< / com:Sub> region fused with a variable domain that is derived from gene segments that consist essentially of a V<com:Sub>L< / com:Sub> gene segment and a J<com:Sub>L< / com:Sub> gene segment, and a second two identical polypeptides that each consists essentially of a C<com:Sub>L< / com:Sub> region fused with a variable domain that is derived from gene segments that consist essentially of a <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment and a <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segment.

[00187] In a specific embodiment, the two identical polypeptides that have the C<com:Sub>H< / com:Sub> region have a mouse C<com:Sub>H< / com:Sub> region.

[88100] In a specific embodiment, the two identical polypeptides that have the C<com:Sub>L< / com:Sub> region have a mouse C<com:Sub>L< / com:Sub> region.

[00189] In one embodiment, the variable domain fused with the C<com:Sub>L< / com:Sub> region is a variable domain that is cognate with the variable domain fused to the C<com:Sub>H< / com:Sub> region.

[00190] In one embodiment, the variable domain that is fused with the endogenous C<com:Sub>H< / com:Sub> region comprises a greater number of somatic hypermutations than the variable domain that is fused with the endogenous C<com:Sub>L< / com:Sub> region; in a specific embodiment, the variable domain fused with the endogenous C<com:Sub>H< / com:Sub> region comprises about 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4- fold, 4.5-fold, or 5-fold or more somatic hypermutations than the variable domain fused to the endogenous C<com:Sub>L< / com:Sub> region. In one embodiment, the variable domain fused with the endogenous C<com:Sub>L< / com:Sub> region is encoded by a gene that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more N additions.

[00191] In one embodiment, one or more of the V segments and the J segments are human gene segments. In a specific embodiment, both the V segments and the J segments are human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segments. In another specific embodiment, both of the V segments and the J segments are human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the V segments and the J segments are independently selected from human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> and human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> gene segments. In a specific embodiment, the V segments are <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segments and the J segments are <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> segments. In another specific embodiment, the V segments are <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segments and the J segments are Jk segments.

[00192] In one embodiment, one or more of the variable domains fused with the C<com:Sub>L< / com:Sub> region and the variable domains fused with the C<com:Sub>H< / com:Sub> region are human variable domains. In a specific embodiment, the human variable domains are human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> domains. In another specific embodiment, the human variable domains are <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> domains. In one embodiment, the human domains are independently selected from human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> domains. In a specific embodiment, the human variable domain fused with the C<com:Sub>L< / com:Sub> region is a human Vλ domain and the human variable domain fused with the C<com:Sub>H< / com:Sub> region is a human V<com:Sub>K< / com:Sub> domain. In another embodiment, the human variable domain fused with the <com:Math><semantics>CL<annotation encoding="application / x-tex">C_L< / annotation>< / semantics>< / com:Math> region is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> domain and the human variable domain fused with the <com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math> is a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> domain.

[00193] In one embodiment, the V<com:Sub>L< / com:Sub> gene segment of the first two identical polypeptides is selected from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment and a human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segment. In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment of the second two identical polypeptides is selected from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment and a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment of the first two identical polypeptides is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment and the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment of the second two identical polypeptides is selected from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment and a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> segment. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment of the first two identical polypeptides is a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment and the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment of the second two identical polypeptides is selected from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment and a human Vκ segment. In a specific embodiment, the human V<com:Sub>L< / com:Sub> segment of the first two identical polypeptides is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment, and the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment of the second two identical polypeptides is a human Vκ segment.

[00194] In one embodiment, the IgG of the mouse comprises a binding protein made in response to an antigen, wherein the binding protein comprises a polypeptide that consists essentially of a variable domain and a C<com:Sub>H< / com:Sub> region, wherein the variable domain is encoded by a nucleotide sequence that consists essentially of a rearranged V<com:Sub>L< / com:Sub> segment and a rearranged J segment, and wherein the binding protein specifically binds an epitope of the antigen with a K<com:Sub>D< / com:Sub> in the micromolar, nanomolar, or picomolar range.

[00195] In one aspect, a mouse is provided, wherein all or substantially all of the IgG made by the mouse in response to an antigen comprises a heavy chain that comprises a variable domain, wherein the variable domain is encoded by a rearranged gene derived from gene segments that consist essentially of a V gene segment and a J gene segment. In one embodiment, the rearranged gene comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00196] In one embodiment, the V segment is a V segment of a light chain. In one embodiment, the light chain is selected from a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain and a <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain. In a specific embodiment, the light chain is a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain. In a specific embodiment, the V segment is a human V segment. In a specific embodiment, the V segment is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment and the J segment is a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segment.

[00197] In one embodiment, the J segment is a J segment of a light chain. In one embodiment, the light chain is selected from a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain and a <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain. In a specific embodiment, the light chain is a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain. In a specific embodiment, the J segment is a human J segment. In another embodiment, the J segment is a J segment of a heavy chain (i.e., a J<com:Sub>H< / com:Sub>). In a specific embodiment, the heavy chain is of mouse origin. In another specific embodiment, the heavy chain is of human origin.

[00198] In one embodiment, the variable domain of the heavy chain that is made from no more than a V segment and a J segment is a somatically mutated variable domain.

[00199] In one embodiment, the variable domain of the heavy chain that is made from no more than a V segment and a J segment is fused to a mouse C<com:Sub>H< / com:Sub> region.

[00200] In a specific embodiment, all or substantially all of the IgG made by the mouse in response to an antigen comprises a variable domain that is derived from no more than one human V segment and no more than one human J segment, and the variable domain is fused to a mouse IgG constant region, and the IgG further comprises a light chain that comprises a human V<com:Sub>L< / com:Sub> domain fused with a mouse C<com:Sub>L< / com:Sub> region. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain fused with the mouse <com:Math><semantics>CL<annotation encoding="application / x-tex">C_L< / annotation>< / semantics>< / com:Math> region is derived from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segment. In a specific embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain fused with the mouse <com:Math><semantics>CL<annotation encoding="application / x-tex">C_L< / annotation>< / semantics>< / com:Math> region is derived from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment and a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> segment.

[00201] In one aspect, a mouse is provided that makes an IgG comprising a first CDR3 on a polypeptide comprising a CH region and a second CDR3 on a polypeptide comprising a C<com:Sub>1< / com:Sub> region, wherein both the first CDR3 and the second CDR3 are each independently derived from no more than two gene segments, wherein the two gene segments consist essentially of a V<com:Sub>L< / com:Sub> gene segment and a J<com:Sub>L< / com:Sub> gene segment. In one embodiment, the CDR3 on the polypeptide comprising the C<com:Sub>H< / com:Sub> region comprises a sequence that is derived from a CDR3 nucleotide sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00202] In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment and the <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segment are human gene segments. In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment and the <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segment are <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> segment and the <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> segment are <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> gene segments.

[00203] In one aspect, a mouse is provided that makes an IgG comprising a first CDR3 on a first polypeptide comprising a C<com:Sub>H< / com:Sub> region and a second CDR3 on a second polypeptide comprising a C<com:Sub>L< / com:Sub> region, wherein both the first CDR3 and the second CDR3 each comprise a sequence of amino acids wherein more than 75% of the amino acids are derived from a V gene segment. In one embodiment, the CDR3 on the polypeptide comprising the CH region comprises a sequence that is derived from a CDR3 nucleotide sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00204] In one embodiment, more than 80%, more than 90%, or more than 95% of the amino acids of the first CDR3, and more than 80%, more than 90%, or more than 95% of the amino acids of the second CDR3, are derived from a light chain V segment.

[00205] In one embodiment, no more than two amino acids of the first CDR3 are derived from a gene segment other than a light chain V segment. In one embodiment, no more than two amino acids of the second CDR3 are derived from a gene segment other than a light chain V segment. In a specific embodiment, no more than two amino acids of the first CDR3 and no more than two amino acids of the second CDR3 are derived from a gene segment other than a light chain V segment. In one embodiment, no CDR3 of the IgG comprises an amino acid sequence derived from a D gene segment. In one embodiment, the CDR3 of the first polypeptide does not comprise a sequence derived from a D segment.

[00206] In one embodiment, the V segment is a human V gene segment. In a specific embodiment, the V segment is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment.

[00207] In one embodiment, the first and / or the second CDR3 have at least one, two, three, four, five, or six somatic hypermutations. In one embodiment, the first CDR3 is encoded by a nucleic acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00208] In one embodiment, the first CDR3 consists essentially of amino acids derived from a human light chain V gene segment and a human light chain J gene segment, and the second CDR3 consists essentially of amino acids derived from a human light chain V gene segment and a human light chain J gene segment. In one embodiment, the first CDR3 is derived from a nucleic acid sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions. In one embodiment, the first CDR3 is derived from no more than two gene segments, wherein the no more than two gene segments are a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment and a human Jκ gene segment; and the second CDR3 is derived from no more than two gene segments, wherein the no more than two gene segments are a human Vk gene segment and a J gene segment selected from a human <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> segment, a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> segment, and a human J<com:Sub>H< / com:Sub> segment. In one embodiment, the first CDR3 is derived from no more than two gene segments, wherein the no more than two gene segments are a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment and a J segment selected from a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segment, a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J_\lambda< / annotation>< / semantics>< / com:Math> segment, and a human J<com:Sub>H< / com:Sub> segment.

[00209] In one aspect, a mouse is provided that makes an IgG that does not contain an amino acid sequence derived from a D<com:Sub>H< / com:Sub> gene segment, wherein the IgG comprises a first polypeptide having a first V<com:Sub>L< / com:Sub> domain fused with a mouse C<com:Sub>L< / com:Sub> region and a second polypeptide having a second V<com:Sub>L< / com:Sub> domain fused with a mouse C<com:Sub>H< / com:Sub> region, wherein the first V<com:Sub>L< / com:Sub> domain and the second V<com:Sub>L< / com:Sub> domain are not identical. In one embodiment, the first and second V<com:Sub>L< / com:Sub> domains are derived from different V segments. In another embodiment, the first and second V<com:Sub>1< / com:Sub> domains are derived from different J segments. In one embodiment, the first and second <com:Math><semantics>VL<annotation encoding="application / x-tex">V_{L}< / annotation>< / semantics>< / com:Math> domains are derived from identical V and J segments, wherein the second <com:Math><semantics>VL<annotation encoding="application / x-tex">V_{L}< / annotation>< / semantics>< / com:Math> domain comprises a higher number of somatic hypermutations as compared to the first <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain.

[00210] In one embodiment, the first and the second V<com:Sub>L< / com:Sub> domains are independently selected from human and mouse <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domains. In one embodiment, the first and second <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domains are independently selected from <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_{\lambda}< / annotation>< / semantics>< / com:Math> domains. In a specific embodiment, the first <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain is selected from a <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> domain and a <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain, and the second <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain is a <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> domain. In another specific embodiment, the <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> domain is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> domain.

[00211] In one aspect, a mouse is provided, wherein all or substantially all of the IgG made by the mouse consists essentially of a light chain having a first human V<com:Sub>L< / com:Sub> domain fused with a mouse C<com:Sub>L< / com:Sub> domain, and a heavy chain having a second human V<com:Sub>L< / com:Sub> domain fused with a mouse C<com:Sub>H< / com:Sub> domain.

[00212] In one embodiment, the human V<com:Sub>L< / com:Sub> domain fused with the mouse C<com:Sub>H< / com:Sub> domain is a human Vκ domain.

[00213] In one embodiment, the first and the second human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domains are not identical.

[00214] In one aspect, a mouse is provided, wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or about 100% of the immunoglobulin G made by the mouse consists essentially of a dimer of (a) a first polypeptide that consists essentially of an immunoglobulin V<com:Sub>L< / com:Sub> domain and an immunoglobulin C<com:Sub>L< / com:Sub> region; and, (b) a second polypeptide of no more than 535 amino acids in length, wherein the second polypeptide consists essentially of a C<com:Sub>H< / com:Sub> region and a V domain that lacks a sequence derived from a D<com:Sub>H< / com:Sub> gene segment.

[00215] In one embodiment, the second polypeptide is about 435-535 amino acids in length. In a specific embodiment, the second polypeptide is about 435-530 amino acids in length. In a specific embodiment, the second polypeptide is about 435-525 amino acids in length. In a specific embodiment, the second polypeptide is about 435-520 amino acids in length. In a specific embodiment, the second polypeptide is about 435-515 amino acids in length.

[00216] In one embodiment, in about 90% or more of the IgG made by the mouse the second polypeptide is no more than about 535 amino acids in length.

[00217] In one embodiment, in about 50% or more of the IgG made by the mouse the second polypeptide is no more than about 535 amino acids in length. In one embodiment, in about 50% or more of the immunoglobulin G made by the mouse the second polypeptide is no more than about 530, 525, 520, 515, 510, 505, 500, 495, 490, 485, 480, 475, 470, 465, 460, 455, or 450 amino acids in length. In one embodiment, about 60%, 70%, 80%, 90%, or 95 % or more of the IgG made by the mouse is of the recited length. In a specific embodiment, all or substantially all of the IgG made by the mouse is of the recited length.

[00218] In one embodiment, the V domain of the second polypeptide is a V<com:Sub>L< / com:Sub> domain. In a specific embodiment, the V domain of the second polypeptide is selected from a <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> and a Vλ domain. In a specific embodiment, the V domain of the second polypeptide is a human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_{\lambda}< / annotation>< / semantics>< / com:Math> domain.

[00219] In one aspect, a mouse is provided that expresses from a nucleotide sequence in its germline a polypeptide that comprises a light chain variable sequence (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, a V and / or J sequence), a D<com:Sub>H< / com:Sub> sequence, and a heavy chain constant region.

[00220] In one embodiment, the mouse expresses the polypeptide from an endogenous heavy chain locus that comprises a replacement of all or substantially all functional endogenous heavy chain variable locus gene segments with a plurality of human gene segments at the endogenous heavy chain locus.

[00221] In one embodiment, the polypeptide comprises a <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> sequence derived from a <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or a <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segment, the polypeptide comprises a CDR3 derived from a <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> gene segment, and the polypeptide comprises a sequence derived from a <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment.

[00222] In one embodiment, the mouse comprises an endogenous heavy chain immunoglobulin locus comprising a replacement of all functional V<com:Sub>H< / com:Sub> gene segments with one or more human light chain <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> gene segments wherein the one or more human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segments each have juxtaposed on the downstream side a 23-mer spaced recombination signal sequence (RSS), wherein the <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segments are operably linked to a human or mouse <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> segment that has juxtaposed upstream and downstream a 12-mer spaced RSS; the D<com:Sub>H< / com:Sub> gene segment is operably linked with a J segment juxtaposed upstream with a 23-mer spaced RSS that is suitable for recombining with the 12-mer spaced RSS juxtaposing the D<com:Sub>H< / com:Sub> gene segment; wherein the V, D<com:Sub>H< / com:Sub>, and J segments are operably linked to a nucleic acid sequence encoding a heavy chain constant region.

[00223] In one embodiment, the mouse comprises an endogenous heavy chain immunoglobulin locus comprising a replacement of all functional V<com:Sub>H< / com:Sub> gene segments with one or more human Vκ gene segments each juxtaposed on the downstream side with a 12-mer spaced recombination signal sequence (RSS), wherein the V segments are operably linked to a human or mouse D<com:Sub>H< / com:Sub> segment that is juxtaposed both upstream and downstream with a 23-mer spaced RSS; the D<com:Sub>H< / com:Sub> segment is operably linked with a J segment juxtaposed on the upstream side with a 12-mer spaced RSS that is suitable for recombining with the 23-mer spaced RSS juxtaposing the D<com:Sub>H< / com:Sub> segment; wherein the V, D<com:Sub>H< / com:Sub>, and gene segments are operably linked to a nucleic acid sequence encoding a heavy chain constant region.

[00224] In one embodiment, the heavy chain constant region is an endogenous heavy chain constant region. In one embodiment, the nucleic acid sequence encodes a sequence selected from a C<com:Sub>H< / com:Sub>1, a hinge, a C<com:Sub>H< / com:Sub>2, a C<com:Sub>H< / com:Sub>3, and a combination thereof. In one embodiment, one or more of the C<com:Sub>H< / com:Sub>1, hinge, C<com:Sub>H< / com:Sub>2, and C<com:Sub>H< / com:Sub>3 are human.

[00225] In one embodiment, the mouse comprises an endogenous heavy chain immunoglobulin locus comprising a replacement of all functional V<com:Sub>H< / com:Sub> gene segments with a plurality of human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> gene segments each juxtaposed downstream with 23-mer spaced RSS, a plurality of human D<com:Sub>H< / com:Sub> segments juxtaposed both upstream and downstream by a 12-mer spaced RSS, a plurality of human J segments (<com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>JR<annotation encoding="application / x-tex">J_R< / annotation>< / semantics>< / com:Math>) juxtaposed both upstream and downstream with a 23-mer spaced RSS, wherein the locus comprises an endogenous constant region sequence selected from C<com:Sub>H< / com:Sub>1, hinge, C<com:Sub>H< / com:Sub>2, C<com:Sub>H< / com:Sub>3, and a combination thereof. In a specific embodiment, the mouse comprises all or substantially all functional human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segments, all or substantially all functional human <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> segments, and all or substantially all <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> segments.

[00226] In one embodiment, the mouse expresses an antigen-binding protein comprising (a) a polypeptide that comprises a human light chain sequence linked to a heavy chain constant sequence comprising a mouse sequence; and (b) a polypeptide that comprises a human light chain variable region linked to a human or mouse light chain constant sequence. In a specific embodiment, the light chain sequence is a human light chain sequence, and upon exposure to a protease that is capable of cleaving an antibody into an Fc and a Fab, a fully human Fab is formed that comprises at least four light chain CDRs, wherein the at least four light chain CDRs are selected from <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> sequences, <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> sequences, and a combination thereof. In one embodiment, the Fab comprises at least five light chain CDRs. In one embodiment, the Fab comprises six light chain CDRs. In one embodiment, at least one CDR of the Fab comprises a sequence derived from a <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment or a <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segment, and the at least one CDR further comprises a sequence derived from a D segment. In one embodiment, the at least one CDR is a CDR3 and the CDR is derived from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> segment, a human D segment, and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segment.

[00227] In one embodiment, the polypeptide of comprises a variable region derived from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> gene segment, a human <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> gene segment, and a human <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segment. In a specific embodiment, the heavy chain constant sequence is derived from a human <com:Math><semantics>CH1<annotation encoding="application / x-tex">C_{H1}< / annotation>< / semantics>< / com:Math> and a mouse <com:Math><semantics>CH2<annotation encoding="application / x-tex">C_{H2}< / annotation>< / semantics>< / com:Math> and a mouse <com:Math><semantics>CH3<annotation encoding="application / x-tex">C_{H3}< / annotation>< / semantics>< / com:Math> sequence.

[00228] In one aspect, a mouse is provided that comprises in its germline an unrearranged human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> gene segment operably linked to a human J gene segment and a heavy chain constant region sequence, wherein the mouse expresses a V<com:Sub>L< / com:Sub> binding protein that comprises a human V<com:Sub>K< / com:Sub> domain fused with a heavy chain constant region and a human V<com:Sub>L< / com:Sub> domain fused with a light chain constant domain. In one embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain comprises a rearranged human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment selected from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> gene segment. In a specific embodiment, the human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain further comprises a rearranged human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segment selected from a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>JR<annotation encoding="application / x-tex">J_R< / annotation>< / semantics>< / com:Math> gene segment.

[00229] In one aspect, a mouse is provided that expresses an immunoglobulin protein from a modified endogenous heavy chain locus in its germline, wherein the modified endogenous heavy chain locus lacks a functional mouse heavy chain V gene segment and the locus comprises unrearranged light chain V gene segments and unrearranged J gene segments, wherein the unrearranged light chain V gene segments and unrearranged J gene segments are operably linked with a heavy chain constant region sequence; wherein the immunoglobulin protein consists essentially of a first polypeptide and a second polypeptide, wherein the first polypeptide comprises an immunoglobulin light chain sequence and an immunoglobulin heavy chain constant sequence, and the second polypeptide comprises an immunoglobulin light chain variable domain and a light chain constant region.

[00230] In one aspect, a mouse is provided that expresses an immunoglobulin protein, wherein the immunoglobulin protein lacks a heavy chain immunoglobulin variable domain, and the immunoglobulin protein comprises a first variable domain derived from a light chain gene, and a second variable domain derived from a light chain gene, wherein the first variable domain and the second variable domain are cognate with respect to one another, wherein the first and the second light chain variable domains are not identical, and wherein the first and the second light chain variable domains associate and when associated specifically bind an antigen of interest.

[00231] In one aspect, a mouse is provided that expresses from unrearranged gene segments in its germline an immunoglobulin protein comprising variable regions that are wholly derived from gene segments that consist essentially of unrearranged human gene segments, wherein the immunoglobulin protein comprises an immunoglobulin light chain constant sequence and an immunoglobulin heavy chain constant sequence selected from the group consisting of a <com:Math><semantics>CH1<annotation encoding="application / x-tex">C_H1< / annotation>< / semantics>< / com:Math>, a hinge, a <com:Math><semantics>CH2<annotation encoding="application / x-tex">C_H2< / annotation>< / semantics>< / com:Math>, a <com:Math><semantics>CH3<annotation encoding="application / x-tex">C_H3< / annotation>< / semantics>< / com:Math>, and a combination thereof.

[00232] In one aspect, a mouse is provided that expresses from unrearranged gene segments in its germline an immunoglobulin protein comprising variable regions, wherein all CDR3s of all variable regions are generated entirely from light chain V and J gene segments, and optionally one or more somatic hypermutations, e.g., one or more N additions.

[00233] In one aspect, a mouse is provided that expresses a somatically mutated immunoglobulin protein derived from unrearranged human immunoglobulin light chain variable region gene segments in the germline of the mouse, wherein the immunoglobulin protein lacks a CDR that comprises a sequence derived from a D gene segment, wherein the immunoglobulin protein comprises a first CDR3 on a light chain variable domain fused with a light chain constant region, comprises a second CDR3 on a light chain variable domain fused with a heavy chain constant region, and wherein the second CDR3 is derived from a rearranged light chain variable region sequence that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more N additions.

[00234] In one aspect, a mouse as described herein is provided, wherein the mouse comprises a functionally silenced light chain locus selected from a <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> locus, a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> locus, and a combination thereof. In one embodiment, the mouse comprises a deletion of a <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> and / or a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> locus, in whole or in part, such that the <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> locus is nonfunctional.

[00235] In one aspect, a mouse embryo is provided, comprising a cell that comprises a modified immunoglobulin locus as described herein. In one embodiment, the mouse is a chimera and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the cells of the embryo comprise a modified immunoglobulin locus as described herein. In one embodiment, at least 96%. 97%, 98%, 99%, or 99.8% of the cells of the embryo comprise a modified immunoglobulin locus as described herein. In one embodiment, the embryo comprises a host cell and a cell derived from a donor ES cell, wherein the cell derived from the donor ES cell comprises a modified immunoglobulin locus as described herein. In one embodiment, the embryo is a 2-, 4-, 8, 16-, 32, or 64-cell stage host embryo, or a blastocyst, and further comprises a donor ES cell comprising a modified immunoglobulin locus as described herein.

[00236] In one aspect, a mouse or a cell made using a nucleic acid construct as described herein is provided.

[00237] In one aspect, a mouse made using a cell as described herein is provided. In one embodiment, the cell is a mouse ES cell.

[00238] In one aspect, mouse cells and mouse embryos are provided, including but not limited to ES cells, pluripotent cells, and induced pluripotent cells, that comprise genetic modifications as described herein. Cells that are XX and cells that are XY are provided. Cells that comprise a nucleus containing a modification as described herein are also provided, e.g., a modification introduced into a cell by pronuclear injection. Cells, embryos, and mice that comprise a virally introduced ADAM6 gene are also provided, e.g., cells, embryos, and mice comprising a transduction construct comprising an ADAM6 gene that is functional in the mouse.

[00239] In one aspect, a cell or tissue derived from a mouse as described herein is provided. In one embodiment, the cell or tissue is derived from spleen, lymph node or bone marrow of a mouse as described herein. In one embodiment, the cell is a B cell. In one embodiment the cell is an embryonic stem cell. In one embodiment, the cell is a germ cell.

[00240] In one embodiment, the tissue is selected from connective, muscle, nervous and epithelial tissue. In a specific embodiment, the tissue is reproductive tissue.

[00241] In one embodiment, the cell and / or tissue derived from a mouse as described herein is isolated for use in one or more ex vivo assays. In various embodiments, the one or more ex vivo assays include measurements of physical, thermal, electrical, mechanical or optical properties, a surgical procedure, measurements of interactions of different tissue types, the development of imaging techniques, or a combination thereof.

[00242] In aspect, use of cell or tissue derived from a mouse as described herein to make an antibody is provided. In one aspect, use of a cell or tissue derived from a mouse as described herein to make a hybridoma or quadroma is provided.

[00243] In one aspect, a non-human cell comprising a chromosome or fragment thereof of a non-human animal as described herein. In one embodiment, the non-human cell comprises a nucleus of a non-human animal as described herein. In one embodiment, the non-human cell comprises the chromosome or fragment thereof as the result of a nuclear transfer.

[00244] In one aspect, a nucleus derived from a mouse as described herein is provided. In one embodiment, the nucleus is from a diploid cell that is not a B cell.

[00245] In one aspect, a genetically modified mouse cell is provided, wherein the cell is incapable of expressing a heavy chain comprising rearranged endogenous immunoglobulin heavy chain gene segments, and the cell comprises a functional ADAM6 gene that encodes a mouse ADAM6 protein or functional fragment thereof. In one embodiment, the cell further comprises an insertion of human immunoglobulin light chain gene segments. In a specific embodiment, the human immunoglobulin light chain gene segments are Vκ and / or Jκ gene segments that are operably linked to mouse heavy chain constant regions such that upon rearrangement encode a functional light chain variable domain fused to a mouse heavy chain constant domain.

[00246] In one aspect, a genetically modified mouse cell is provided; wherein the cell lacks a functional endogenous ADAM6 locus, and the cell comprises an ectopic nucleotide sequence that encodes a mouse ADAM6. In one embodiment, the cell further comprises a modification of an endogenous immunoglobulin heavy chain variable region sequence. In a specific embodiment, the modification of the endogenous immunoglobulin heavy chain variable region sequence comprises a deletion selected from a deletion of a mouse V<com:Sub>H< / com:Sub> gene segment, a deletion of a mouse D<com:Sub>H< / com:Sub> gene segment, a deletion of a mouse J<com:Sub>H< / com:Sub> gene segment, and a combination thereof. In a specific embodiment, the mouse comprises a replacement of one or more mouse immunoglobulin V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and / or J<com:Sub>H< / com:Sub> sequences with a human immunoglobulin sequence. In a specific embodiment, the human immunoglobulin sequence is selected from a human <com:Math><semantics>VH<annotation encoding="application / x-tex">V_H< / annotation>< / semantics>< / com:Math>, a human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math>, a human <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math>, a human <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math>, a human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math>, and a combination thereof.

[00247] In one embodiment, the cell is a totipotent cell, a pluripotent cell, or an induced pluripotent cell. In a specific embodiment, the cell is a mouse ES cell.

[00248] In one aspect, a mouse B cell is provided, wherein the mouse B cell comprises a rearranged immunoglobulin gene, wherein the B cell comprises on a chromosome of the B cell a nucleic acid sequence encoding an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the mouse B cell comprises two alleles of the nucleic acid sequence. In one embodiment, the rearranged immunoglobulin gene comprises a rearranged immunoglobulin light chain sequence contiguous with a heavy chain constant sequence. In one embodiment, the light chain sequence is a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> sequence; in one embodiment, the light chain sequence is a <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> sequence.

[00249] In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (e.g., a B cell chromosome) that is contiguous with the rearranged mouse heavy chain immunoglobulin locus.

[00250] In one embodiment, the nucleic acid sequence is on a nucleic acid molecule (e.g., a B cell chromosome) that is distinct from the nucleic acid molecule that comprises the rearranged mouse heavy chain immunoglobulin locus.

[00251] In one embodiment, the mouse B cell comprises a rearranged non-mouse light chain immunoglobulin variable region sequence operably linked to a mouse or human heavy chain immunoglobulin constant region gene, wherein the B cell comprises a nucleic acid sequence that encodes an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a male mouse.

[00252] In one aspect, a somatic mouse cell is provided, comprising a chromosome that comprises a modified immunoglobulin heavy chain locus, and a nucleic acid sequence encoding a mouse ADAM6 or ortholog or homolog or fragment thereof that is functional in a male mouse. In one embodiment, the nucleic acid sequence is on the same chromosome as the modified immunoglobulin heavy chain locus. In one embodiment, the nucleic acid is on a different chromosome than the modified immunoglobulin heavy chain locus. In one embodiment, the somatic cell comprises a single copy of the nucleic acid sequence. In one embodiment, the somatic cell comprises at least two copies of the nucleic acid sequence. In a specific embodiment, the somatic cell is a B cell. In one embodiment, the modified immunoglobulin heavy chain locus comprises an unrearranged immunoglobulin light chain gene segment operably linked to a heavy chain constant region sequence.

[00253] In one aspect, a mouse germ cell is provided, comprising a nucleic acid sequence encoding a mouse ADAM6 (or homolog or ortholog or functional fragment thereof) on a chromosome of the germ cell, wherein the nucleic acid sequence encoding the mouse ADAM6 (or homolog or ortholog or functional fragment thereof) is at a position in the chromosome that is different from a position in a chromosome of a wild-type mouse germ cell, wherein the mouse further comprises a modification comprising an unrearranged light chain immunoglobulin gene segment (a <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J_\lambda< / annotation>< / semantics>< / com:Math>) operably linked to a heavy chain constant region sequence. In one embodiment, the nucleic acid sequence is at a mouse immunoglobulin locus. In one embodiment, the nucleic acid sequence is on the same chromosome of the germ cell as a mouse immunoglobulin locus. In one embodiment, the nucleic acid sequence is on a different chromosome of the germ cell than the mouse immunoglobulin locus. In one embodiment, the mouse immunoglobulin locus comprises a replacement of at least one mouse immunoglobulin sequence with at least one non-mouse immunoglobulin sequence. In a specific embodiment, the at least one non-mouse immunoglobulin sequence is a human immunoglobulin sequence.

[00254] In one aspect, a pluripotent, induced pluripotent, or totipotent cell derived from a mouse as described herein is provided. In a specific embodiment, the cell is a mouse embryonic stem (ES) cell.

[00255] In one aspect, a cell is provided, comprising a modified immunoglobulin locus as described herein. In one embodiment, the cell is selected from a totipotent cell, a pluripotent cell, an induced pluripotent stem cell (iPS), and an ES cell. In a specific embodiment, the cell is a mouse cell, e.g., a mouse ES cell. In one embodiment, the cell is homozygous for the modified immunoglobulin locus.

[00256] In one aspect, a cell is provided, comprising a nucleic acid sequence encoding a first polypeptide that comprises a first somatically mutated human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> sequence fused to a human heavy chain constant region sequence.

[00257] In one embodiment, the cell further comprises a second polypeptide chain that comprises a second somatically mutated human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> sequence fused to a human light chain constant region sequence.

[00258] In one embodiment, the human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> sequence of the first polypeptide is cognate with the human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_{\lambda}< / annotation>< / semantics>< / com:Math> sequence of the second polypeptide.

[00259] In one embodiment, the <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> of the first polypeptide and the human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> of the second polypeptide when associated specifically bind an antigen of interest. In a specific embodiment, the first polypeptide comprises a variable domain consisting essentially of a human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V_{\kappa}< / annotation>< / semantics>< / com:Math>, and the second polypeptide comprises a variable domain consisting of a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> that is cognate with the human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> of the first polypeptide, and the human constant region sequence is an IgG sequence.

[00260] In one embodiment, the cell is selected from a CHO cell, a COS cell, a 293 cell, a HeLa cell, and a human retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell.

[00261] In one aspect, a somatic rodent (e.g., mouse) cell is provided, comprising a chromosome that comprises a genetic modification as described herein.

[00262] In one aspect, a rodent (e.g., mouse) germ cell is provided, comprising a nucleic acid sequence that comprises a genetic modification as described herein.

[00263] In one aspect, a pluripotent, induced pluripotent, or totipotent cell derived from a rodent (e.g., mouse) as described herein is provided. In a specific embodiment, the cell is a mouse embryonic stem (ES) cell.

[00264] In one aspect, use of a cell as described herein for the manufacture of a rodent <com:Math><semantics>(e.g.,amouse)<annotation encoding="application / x-tex">(e.g., a mouse)< / annotation>< / semantics>< / com:Math>, a cell, or a therapeutic protein <com:Math><semantics>(e.g.,anantibodyorotherantigen−binding)<annotation encoding="application / x-tex">(e.g., an antibody or other antigen-binding)< / annotation>< / semantics>< / com:Math> protein) is provided. In one embodiment, use of a cell as described herein for the manufacture of a therapeutic protein is provided, wherein the therapeutic protein comprises a human variable domain. In a specific embodiment, the human variable domain comprises rearranged <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments.

[00265] In one aspect, a rodent (e.g., a mouse) made using a targeting vector, nucleotide construct, or cell as described herein is provided.

[00266] In one aspect, use of a targeting vector as described herein for the manufacture of a rodent (e.g., a mouse) or a cell (e.g., a mouse ES cell, a mouse fibroblast, etc.) is provided. In one embodiment, the targeting vector comprises a human genomic fragment that contains unrearranged immunoglobulin gene segments. In a specific embodiment, the unrearranged immunoglobulin gene segments include V and J gene segments. In a specific embodiment, the unrearranged immunoglobulin gene segments include V, D and J gene segments.

[00267] In one aspect, a nucleic acid construct is provided, comprising an upstream homology arm and a downstream homology arm, wherein the upstream homology arm comprises a sequence that is identical or substantially identical to a human immunoglobulin heavy chain variable region sequence, the downstream homology arm comprises a sequence that is identical or substantially identical to a human or mouse immunoglobuling variable region sequence, and disposed between the upstream and downstream homology arms is a sequence that comprises a nucleotide sequence encoding a mouse ADAM6 protein. In a specific embodiment, the sequence encoding the mouse ADAM6 gene is operably linked with a mouse promoter with which the mouse ADAM6 is linked in a wild type mouse.

[00268] In one aspect, a targeting vector is provided, comprising (a) a nucleotide sequence that is identical or substantially identical to a human variable region gene segment nucleotide sequence; and, (b) a nucleotide sequence encoding a mouse ADAM6 or ortholog or homolog or fragment thereof that is functional in a mouse.

[00269] In one embodiment, the targeting vector further comprises a promoter operably linked to the sequence encoding the mouse ADAM6. In a specific embodiment, the promoter is a mouse ADAM6 promoter.

[00270] In one aspect, a nucleotide construct for modifying a mouse immunoglobulin heavy chain variable locus is provided, wherein the construct comprises at least one site- specific recombinase recognition site and a sequence encoding an ADAM6 protein or ortholog or homolog or fragment thereof that is functional in a mouse.

[00271] In one aspect, a nucleic acid construct is provided that comprises a human D<com:Sub>H< / com:Sub> gene segment juxtaposed upstream and downstream with a 23-mer spaced RSS. In a specific embodiment, the nucleic acid construct comprises a homology arm that is homologous to a human genomic sequence comprising human Vk gene segments. In one embodiment, the targeting construct comprises all or substantially all human D<com:Sub>H< / com:Sub> gene segments each juxtaposed upstream and downstream with a 23-mer spaced RSS.

[00272] In one aspect, a nucleic acid construct is provided that comprises a human <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segment juxtaposed upstream with a 12-mer spaced RSS. In a specific embodiment, the nucleic acid construct comprises a first homology arm that contains homology to a human genomic D<com:Sub>H< / com:Sub> gene sequence that is juxtaposed upstream and downstream with a 23- mer spaced RSS. In one embodiment, the nucleic acid construct comprises a second homology arm that contains homology to a human genomic J gene sequence or that contains homology to a mouse heavy chain constant region sequence or that contains homology to a J-C intergenic sequence upstream of a mouse constant region heavy chain sequence.

[00273] In one aspect, a nucleic acid construct is provided that comprises a human Vλ segment juxtaposed downstream with a 23-mer spaced RSS, a human D<com:Sub>H< / com:Sub> segment juxtaposed upstream and downstream with a 12-mer spaced RSS, and a human J segment selected from a <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segment juxtaposed upstream with a 23-mer spaced RSS, a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segment juxtaposed upstream with a 23-mer spaced RSS, and a human J<com:Sub>H< / com:Sub> segment juxtaposed upstream with a 23-mer spaced RSS. In one embodiment, the construct comprises a homology arm that contains homology to a mouse constant region sequence, a J-C intergenic mouse sequence, and / or a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> sequence.

[00274] In one embodiment, the nucleic acid construct comprises a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence that comprises a fragment of cluster A of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus. In a specific embodiment, the fragment of cluster A of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus extends from <com:Math><semantics>hVλ3−27<annotation encoding="application / x-tex">hV\lambda 3-27< / annotation>< / semantics>< / com:Math> through <com:Math><semantics>hVλ3−1<annotation encoding="application / x-tex">hV\lambda 3-1< / annotation>< / semantics>< / com:Math>.

[00275] In one embodiment, the nucleic acid construct comprises a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence that comprises a fragment of cluster B of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus. In a specific embodiment, the fragment of cluster B of the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain locus extends from <com:Math><semantics>hVλ5−52<annotation encoding="application / x-tex">hV\lambda 5-52< / annotation>< / semantics>< / com:Math> through <com:Math><semantics>hVλ1−40<annotation encoding="application / x-tex">hV\lambda 1-40< / annotation>< / semantics>< / com:Math>.

[00276] In one embodiment, nucleic acid construct comprises a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence that comprises a genomic fragment of cluster A and a genomic fragment of cluster B. In a one embodiment, the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence comprises at least one gene segment of cluster A and at least one gene segment of cluster B.

[00277] In one embodiment, the human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence comprises at least one gene segment of cluster B and at least one gene segment of cluster C.

[00278] In one aspect, a nucleic acid construct is provided, comprising a human D<com:Sub>H< / com:Sub> segment juxtaposed upstream and downstream with a 23-mer spaced RSS normally found in nature flanking either a <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math>, a <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math>, a <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math>, or a <com:Math><semantics>VH<annotation encoding="application / x-tex">V_H< / annotation>< / semantics>< / com:Math> segment. In one embodiment, the nucleic acid construct comprises a first homology arm homologous to a human V-J intergenic region or homologous to a human genomic sequence comprising a human V gene segment. In one embodiment, the nucleic acid construct comprises a second homology arm homologous to a human or mouse heavy chain constant region sequence. In a specific embodiment, the human or mouse heavy chain constant region sequence is selected from a C<com:Sub>H< / com:Sub>1, hinge, C<com:Sub>H< / com:Sub>2, C<com:Sub>H< / com:Sub>3, and a combination thereof. In one embodiment, the nucleic acid construct comprises a human J gene segment flanked upstream with a 12-mer RSS. In one embodiment, the nucleic acid construct comprises a second homology arm that contains homology to a J gene segment flanked upstream with a 12-mer RSS. In one embodiment, the J gene segment is selected from a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math>, a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J_\lambda< / annotation>< / semantics>< / com:Math>, and a human <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math>.

[00279] In one aspect, a nucleic acid construct is provided that comprises a human D<com:Sub>H< / com:Sub> segment juxtaposed upstream and downstream with a 23-mer spaced RSS, and a site- specific recombinase recognition sequence, e.g., a sequence recognized by a site-specific recombinase such as a Cre, a Flp, or a Dre protein.

[00280] In one aspect, a nucleic acid construct is provided that comprises a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or a human V<com:Sub>K< / com:Sub> segment, a D<com:Sub>H< / com:Sub> segment juxtaposed upstream and downstream with a 12-mer or a 23-mer spaced RSS, and a human J segment with a 12-mer or a 23-mer spaced RSS, wherein the 12-mer or 23-mer spaced RSS is positioned immediately 5' to the human J segment (i.e., with respect to the direction of transcription). In one embodiment, the construct comprises a human Vλ juxtaposed with a 3' 23-mer spaced RSS, a human D<com:Sub>H< / com:Sub> segment juxtaposed upstream and downstream with a 12-mer spaced RSS, and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> segment juxtaposed with a 5' 23-mer spaced RSS. In one embodiment, the construct comprises a human Vκ juxtaposed with a 3' 12-mer spaced RSS, a human D<com:Sub>H< / com:Sub> segment juxtaposed upstream and downstream with a 23-mer spaced RSS, and a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> segment juxtaposed with a 5' 12-mer spaced RSS.

[00281] In one aspect, a targeting vector is provided, comprising (a) a first targeting arm and a second targeting arm, wherein the first and second targeting arms are independently selected from human and mouse targeting arms, wherein the targeting arms direct the vector to an endogenous or modified immunoglobulin V region gene locus; and, (b) a contiguous sequence of human V<com:Sub>L< / com:Sub> gene segments or a contiguous sequence of human V<com:Sub>L< / com:Sub> gene segments and at least one human Jk gene segment, wherein the contiguous sequence is selected from the group consisting of (i) <com:Math><semantics>hVκ4−1<annotation encoding="application / x-tex">hV\kappa 4-1< / annotation>< / semantics>< / com:Math> through <com:Math><semantics>hVκ1−6<annotation encoding="application / x-tex">hV\kappa 1-6< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Jκ1<annotation encoding="application / x-tex">J\kappa 1< / annotation>< / semantics>< / com:Math>, (ii) <com:Math><semantics>hVκ4−1<annotation encoding="application / x-tex">hV\kappa 4-1< / annotation>< / semantics>< / com:Math> through hV<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>1-6 and J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>1 through J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>2, (iii) hV<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>4-1 through hV<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>1-6 and J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>1 through J<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>3, (iv) hVκ4-1 through hVκ1-6 and Jκ1 through Jκ4, (v) hVκ4-1 through hVκ1-6 and Jκ1 through Jκ5, (vi) hVκ3-7 through hVκ1-16, (vii) hVκ1-17 through hVκ2-30, (viii) hVκ3-31 through <com:Math><semantics>hVκ2−40<annotation encoding="application / x-tex">hV\kappa 2-40< / annotation>< / semantics>< / com:Math>, and (ix) a combination thereof.

[00282] In one embodiment, the targeting arms that direct the vector to an endogenous or modified immunoglobulin locus are identical or substantially identical to a sequence at the endogenous or modified immunoglobulin locus.

[00283] In one aspect, a method for making a genetically modified mouse is provided, comprising replacing one or more immunoglobulin heavy chain gene segments upstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of an endogenous ADAM6 locus of the mouse with one or more human immunoglobulin light chain and / or heavy chain gene segments, and replacing one or more immunoglobulin gene segments downstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of the ADAM6 locus of the mouse with one or more human immunoglobuling heavy chain or light chain gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAM6 locus of the mouse include V gene segments. In one embodiment, the human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAM6 locus of the mouse include V and D gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments downstream of an endogenous ADAM6 locus of the mouse include J gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments downstream of an endogenous ADAM6 locus of the mouse include D and J gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments downstream of an endogenous ADAM6 locus of the mouse include V, D and J gene segments.

[00284] In one aspect, a method for making a genetically modified mouse is provided, comprising replacing one or more immunoglobulin heavy chain gene segments upstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of an endogenous ADAM6 locus of the mouse with one or more human immunoglobulin light chain gene segments, and replacing one or more immunoglobulin gene segments downstream (with respect to transcription of the immunoglobulin heavy chain gene segments) of the ADAM6 locus of the mouse with one or more human immunoglobulin light chain segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAM6 locus of the mouse include V gene segments. In one embodiment, the human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments upstream of an endogenous ADAM6 locus of the mouse include V and J gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments downstream of an endogenous ADAM6 locus of the mouse include J gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments downstream of an endogenous ADAM6 locus of the mouse include V and J gene segments. In one embodiment, the one or more human immunoglobulin gene segments replacing one or more endogenous immunoglobulin gene segments downstream of an endogenous ADAM6 locus of the mouse include V gene segments.

[00285] In a specific embodiment, the V gene segments are V<com:Sub>L< / com:Sub> gene segments. In another specific embodiment, the J gene segments are J<com:Sub>L< / com:Sub> gene segments.

[00286] In one embodiment, the one or more immunoglobulin heavy chain gene segments upstream and / or downstream of the ADAM6 gene are replaced in a pluripotent, induced pluripotent, or totipotent cell to form a genetically modified progenitor cell; the genetically modified progenitor cell is introduced into a host; and, the host comprising the genetically modified progenitor cell is gestated to form a mouse comprising a genome derived from the genetically modified progenitor cell. In one embodiment, the host is an embryo. In a specific embodiment, the host is selected from a mouse pre-morula (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, 8- or 4-cell stage), a tetraploid embryo, an aggregate of embryonic cells, or a blastocyst.

[00287] In one aspect, a method for making a genetically modified mouse is provided, comprising replacing a mouse nucleotide sequence that comprises a mouse immunoglobulin gene segment and a mouse ADAM6 (or ortholog or homolog or fragment thereof functional in a male mouse) nucleotide sequence with a sequence comprising a human immunoglobulin gene segment to form a first chimeric locus, then inserting a sequence comprising a mouse ADAM6-encoding sequence (or a sequence encoding an ortholog or homolog or functional fragment thereof) into the sequence comprising the human immunoglobulin gene segment to form a second chimeric locus.

[00288] In one embodiment, the second chimeric locus comprises a human immunoglobulin heavy chain variable gene segment (V<com:Sub>H< / com:Sub>). In one embodiment, the second chimeric locus comprises a human immunoglobulin light chain variable gene segment (V<com:Sub>L< / com:Sub>). In a specific embodiment, the second chimeric locus comprises a human V<com:Sub>H< / com:Sub> gene segment or a human V<com:Sub>L< / com:Sub> gene segment operably linked to a human D<com:Sub>H< / com:Sub> gene segment and a human J<com:Sub>H< / com:Sub> gene segment. In a specific embodiment, the second chimeric locus comprises a human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> gene segment operably linked to a human J<com:Sub>H< / com:Sub> or a human J<com:Sub>L< / com:Sub> gene segment. In a further specific embodiment, the second chimeric locus is operably linked to a third chimeric locus that comprises a human C<com:Sub>H< / com:Sub>1 sequence, or a human C<com:Sub>H< / com:Sub>1 and human hinge sequence, fused with a mouse <com:Math><semantics>CH2+CH3<annotation encoding="application / x-tex">C_{H}2 + C_{H}3< / annotation>< / semantics>< / com:Math> sequence.

[00289] In one aspect, a method is provided for modifying a heavy chain immunoglobulin locus of a mouse, comprising: (a) making a first modification of a mouse heavy chain immunoglobulin locus that results in a reduction or elimination of endogenous mouse ADAM6 activity in a male mouse; and, (b) making a second modification to add a nucleic acid sequence that confers upon the mouse ADAM6 activity that is functional in a male mouse.

[00290] In one embodiment, the first modification comprises the addition of a human immunoglobulin sequence or the replacement of a mouse immunoglobulin sequence with a human immunoglobulin sequence.

[00291] In one embodiment, the human immunoglobulin sequence is a heavy chain sequence. In one embodiment, the human immunoglobulin sequence is a light chain sequence.

[00292] In one embodiment, the first and the second modification are made in a single ES cell, and the single ES cell is introduced into a host embryo to make the mouse.

[00293] In one aspect, a progeny of a mating of a mouse as described herein with a second mouse that is a wild-type mouse or genetically modified is provided.

[00294] In one aspect, use of a mouse that comprises an ectopic nucleotide sequence comprising a mouse ADAM6 locus or sequence to make a fertile male mouse is provided, wherein the use comprises mating the mouse comprising the ectopic nucleotide sequence that comprises the mouse ADAM6 locus or sequence to a mouse that lacks a functional endogenous ADAM6 locus or sequence, and obtaining a progeny that is a female capable of producing progeny having the ectopic ADAM6 locus or sequence or that is a male that comprises the ectopic ADAM6 locus or sequence, and the male exhibits a fertility that is approximately the same as a fertility exhibited by a wild-type male mouse.

[00295] In one aspect, use of a mouse as described herein to introduce an ectopic ADAM6 sequence into a mouse that lacks a functional endogenous ADAM6 sequence is provided, wherein the use comprises mating a mouse as described herein with the mouse that lacks the functional endogenous ADAM6 sequence.

[00296] In one aspect, use of genetic material from a mouse as described herein to make a mouse having an ectopic ADAM6 sequence is provided. In one embodiment, the use comprises nuclear transfer using a nucleus of a cell of a mouse as described herein. In one embodiment, the use comprises cloning a cell of a mouse as described herein to produce an animal derived from the cell. In one embodiment, the use comprises employing a sperm or an egg of a mouse as described herein in a process for making a mouse comprising the ectopic ADAM6 sequence.

[00297] In one aspect, a method for making a fertile male mouse comprising a modified immunoglobulin heavy chain locus is provided, comprising fertilizing a first mouse germ cell that comprises a modification of an endogenous heavy chain immunoglobulin locus with a second mouse germ cell that comprises an ADAM6 gene or ortholog or homolog or fragment thereof that is functional in a male mouse; forming a fertilized cell; allowing the fertilized cell to develop into an embryo; and, gestating the embryo in a surrogate to obtain a mouse.

[00298] In one embodiment, the fertilization is achieved by mating a male mouse and a female mouse. In one embodiment, the female mouse comprises the ADAM6 gene or ortholog or homolog or fragment thereof. In one embodiment, the male mouse comprises the ADAM6 gene or ortholog or homolog or fragment thereof.

[00299] In one aspect, use of a nucleic acid sequence encoding a mouse ADAM6 protein or an ortholog or homolog thereof or a functional fragment of the corresponding ADAM6 protein for restoring or enhancing the fertility of a mouse having a genome comprising a modification of an immunoglobulin heavy chain locus is provided, wherein the modification reduces or eliminates endogenous ADAM6 function.

[00300] In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at an ectopic position. In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at an endogenous immunoglobulin locus. In a specific embodiment, the endogenous immunoglobulin locus is a heavy chain locus. In one embodiment, the nucleic acid sequence is integrated into the genome of the mouse at a position other than an endogenous immunoglobulin locus.

[00301] In one aspect, a method for making a genetically modified mouse is provided, comprising replacing at an endogenous heavy chain locus one or more immunoglobulin heavy chain gene segments of a mouse with one or more human immunoglobulin light chain gene segments. In one embodiment, the replacement is of all or substantially all functional mouse immunoglobulin heavy chain segments (i.e., V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and J<com:Sub>H< / com:Sub> segments) with one or more functional human light chain segments (i.e., V<com:Sub>L< / com:Sub> and J<com:Sub>L< / com:Sub> segments). In one embodiment, the replacement is of all or substantially all functional mouse heavy chain V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and J<com:Sub>H< / com:Sub> segments with all or substantially all human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segments and at least one <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> segment. In a specific embodiment, the replacement includes all or substantially all functional human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> segments.

[00302] In one aspect, a method is provided for making a mouse that expresses a polypeptide that comprises a sequence derived from a human immunoglobulin <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> segment fused with a mouse heavy chain constant region, comprising replacing endogenous heavy chain immunoglobulin variable segments (V<com:Sub>H< / com:Sub>, D<com:Sub>H< / com:Sub>, and J<com:Sub>H< / com:Sub>) with at least one human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segment and at least one human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> segment, wherein the replacement is in a pluripotent, induced pluripotent, or totipotent mouse cell to form a genetically modified mouse progenitor cell; the genetically modified mouse progenitor cell is introduced into a mouse host; and, the mouse host comprising the genetically modified progenitor cell is gestated to form a mouse comprising a genome derived from the genetically modified mouse progenitor cell. In one embodiment, the host is an embryo. In a specific embodiment, the host is selected from a mouse pre-morula (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, 8- or 4-cell stage), a tetraploid embryo, an aggregate of embryonic cells, or a blastocyst.

[00303] In one aspect, a method is provided for making a genetically modified mouse as described herein, comprising introducing by nuclear transfer a nucleic acid containing a modification as described herein into a cell, and maintaining the cell under suitable conditions (e.g., including culturing the cell and gestating an embryo comprising the cell in a surrogate mother) to develop into a mouse as described herein.

[00304] In one aspect, a method for making a modified mouse is provided, comprising modifying as described herein a mouse ES cell or pluripotent or totipotent or induced pluripotent mouse cell to include one or more unrearranged immunoglobulin light chain variable gene segments operably linked to an immunoglobulin heavy chain constant sequence, culturing the ES cell, introducing the cultured ES cell into a host embryo to form a chimeric embryo, and introducing the chimeric embryo into a suitable host mouse to develop into a modified mouse. In one embodiment, the one or more unrearranged immunoglobuling light chain variable region gene segments are human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> or human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the one or more unrearranged immunoglobulin light chain variable region gene segments comprise human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segments and one or more <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math>, or <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> segments. In one embodiment, the heavy chain constant gene sequence is a human sequence selected from C<com:Sub>H< / com:Sub>1, hinge, C<com:Sub>H< / com:Sub>2, C<com:Sub>H< / com:Sub>3, and a combination thereof. In one embodiment, the one or more unrearranged immunoglobulin light chain variable gene segments replace all or substantially all functional endogenous heavy chain variable region gene segments at the endogenous heavy chain locus, and the heavy chain constant sequence is a mouse sequence comprising a <com:Math><semantics>CH1<annotation encoding="application / x-tex">C_H1< / annotation>< / semantics>< / com:Math>, a hinge, a <com:Math><semantics>CH2<annotation encoding="application / x-tex">C_H2< / annotation>< / semantics>< / com:Math>, and a <com:Math><semantics>CH3<annotation encoding="application / x-tex">C_H3< / annotation>< / semantics>< / com:Math>.

[00305] In one aspect, nucleic acids constructs, cells, embryos, mice, and methods are provided for making proteins that comprise one or more <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region immunoglobulin sequences and an immunoglobulin heavy chain constant region sequence, including proteins that comprise a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable domain and a human or mouse heavy chain constant region sequence.

[00306] In one aspect, a nucleotide sequence encoding an immunoglobulin variable region made in a mouse as described herein is provided.

[00307] In one aspect, a heavy chain or light chain variable region amino acid sequence of an antibody made in a mouse as described herein is provided.

[00308] In one aspect, a heavy chain or light chain variable region nucleotide sequence encoding a variable region of an antibody made in a mouse as described herein is provided.

[00309] In one aspect, an antibody or antigen-binding fragment thereof (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, Fab, F(ab)<com:Sub>2< / com:Sub>, scFv) made in a mouse as described herein is provided.

[00310] In one aspect, binding proteins are described that comprise immunoglobulin variable domains that are derived from light chain (i.e., kappa (<com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math>) and / or lambda (<com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math>)) immunoglobulin variable domains, but not from full-length heavy chain immunoglobulin variable domains. Methods and compositions for making binding proteins, including genetically modified mice, are also provided.

[00311] In one aspect, a method is provided for making an antigen-binding protein that does not comprise an immunoglobulin heavy chain variable domain, comprising a step of immunizing a mouse as described herein with an antigen of interest, and maintaining the mouse under conditions that allow it to make an antigen-binding protein that specifically binds the antigen of interest.

[00312] In one aspect, a method is provided for making an antigen-binding protein that comprises a first immunoglobulin light chain variable domain contiguous with a heavy chain constant regions sequence, and a second immunoglobulin light chain variable domain that is contiguous with a light chain constant sequence, comprising a step of immunizing a mouse as described herein with an antigen of interest, and maintaining the mouse under conditions that allow it to make an antigen-binding protein that specifically binds the antigen of interest.

[00313] In one aspect, a method is provided for making an antigen-binding protein that comprises an immunoglobulin light chain variable domain contiguous with a heavy chain constant regions sequence, wherein the antigen-binding protein lacks an immunoglobulin light chain comprising a step of immunizing a mouse as described herein that comprises a knockout of an endogenous <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> locus (or that comprises a nonfunctional endogenous <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> and / or <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> locus) with an antigen of interest, and maintaining the mouse under conditions that allow it to make an antigen-binding protein that specifically binds the antigen of interest.

[00314] In one aspect, use of a mouse as described herein to make an immunoglobulin variable region nucleotide sequence is provided. In one embodiment, the variable region nucleotide sequence comprises rearranged V<com:Sub>L< / com:Sub>, D<com:Sub>H< / com:Sub> and J<com:Sub>L< / com:Sub> gene segments. In one embodiment, the variable region nucleotide sequence comprises rearranged V<com:Sub>L< / com:Sub>, D<com:Sub>H< / com:Sub> and J<com:Sub>H< / com:Sub> gene segments. In one embodiment, the variable region nucleotide sequence comprises rearranged <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the variable region nucleotide sequence comprises rearranged <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segments.

[00315] In one aspect, use of a mouse as described herein to make a fully human Fab or a fully human F(ab)<com:Sub>2< / com:Sub> is provided. In one embodiment, the fully human Fab or a fully human <com:Math><semantics>F(ab)2<annotation encoding="application / x-tex">F(ab)_2< / annotation>< / semantics>< / com:Math> comprises rearranged <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math>, <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the fully human Fab or a fully human F(ab)<com:Sub>2< / com:Sub> comprises rearranged V<com:Sub>L< / com:Sub>, D<com:Sub>H< / com:Sub> and J<com:Sub>H< / com:Sub> gene segments. In one embodiment, the fully human Fab or a fully human <com:Math><semantics>F(ab)2<annotation encoding="application / x-tex">F(ab)_2< / annotation>< / semantics>< / com:Math> comprises rearranged <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments. In one embodiment, the fully human Fab or a fully human F(ab)<com:Sub>2< / com:Sub> comprises rearranged <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segments.

[00316] In one aspect, use of a mouse as described herein to make a fully human Fab (comprising a first human V<com:Sub>L< / com:Sub> fused with a human light chain constant region, and a second human V<com:Sub>L< / com:Sub> fused with a human heavy chain constant region sequence) or a fully human <com:Math><semantics>F(ab)2<annotation encoding="application / x-tex">F(ab)_2< / annotation>< / semantics>< / com:Math> is provided.

[00317] In one aspect, use of a mouse as described herein to make an immortalized cell line is provided. In one embodiment, the immortalized cell line comprises a nucleic acid sequence encoding a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> domain operably linked to a nucleic acid sequence that comprises a mouse constant region nucleic acid sequence. In one embodiment, the immortalized cell line expresses an antibody that comprises a human variable domain. In a specific embodiment, the human variable domain is a light chain variable domain.

[00318] In one aspect, use of a mouse as described herein to make a hybridoma or quadroma is provided. In one embodiment, the hybridoma or quadroma expresses a polypeptide comprising a human variable domain that binds an antigen of interest.

[00319] In one aspect, use of a mouse as described herein to make a phage library containing human light chain variable regions is provided. In one embodiment, the light chain variable regions are human Vκ regions.

[00320] In one aspect, use of a mouse as described herein to generate a variable region sequence for making a human antigen-binding protein is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating a lymphocyte from the immunized mouse of (a), (c) exposing the lymphocyte to one or more labeled antibodies, (d) identifying a lymphocyte that is capable of binding to the antigen of interest, and (e) amplifying one or more human light chain variable region nucleic acid sequences from the lymphocyte thereby generating a variable region sequence.

[00321] In one embodiment, the lymphocyte is derived from the spleen of the mouse. In one embodiment, the lymphocyte is derived from a lymph node of the mouse. In one embodiment, the lymphocyte is derived from the bone marrow of the mouse.

[00322] In one embodiment, the labeled antibody is a fluorophore-conjugated antibody. In one embodiment, the one or more fluorophore-conjugated antibodies are selected from an IgM, an IgG, and / or a combination thereof.

[00323] In one embodiment, the lymphocyte is a B cell.

[00324] In one embodiment, the one or more variable region nucleic acid sequences comprises a light chain variable region sequence. In a specific embodiment, the light chain variable region sequence is an immunoglobulin <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence. In one embodiment, the one or more variable region nucleic acid sequence is a <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain variable region sequence.

[00325] In one embodiment, use of a mouse as described herein to generate one or more <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequences for making a human antigen-binding protein is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating the spleen from the immunized mouse of (a), (c) exposing B lymphocytes from the spleen to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region nucleic acid sequence from the B lymphocyte thereby generating the <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence.

[00326] In one embodiment, use of a mouse as described herein to generate a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence for making a human antigen-binding protein is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating one or more lymph nodes from the immunized mouse of (a), (c) exposing B lymphocytes from the one or more lymph nodes to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a k light chain variable region nucleic acid sequence from the B lymphocyte thereby generating <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence.

[00327] In one embodiment, use of a mouse as described herein to generate a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence for making a human antigen-binding protein is provided, comprising (a) immunizing a mouse as described herein with an antigen of interest, (b) isolating bone marrow from the immunized mouse of (a), (c) exposing B lymphocytes from the bone marrow to one or more labeled antibodies, (d) identifying a B lymphocyte of (c) that is capable of binding to the antigen of interest, and (e) amplifying a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region nucleic acid sequence from the B lymphocyte thereby generating the κ light chain variable region sequence. In various embodiments, the one or more labeled antibodies are selected from an IgM, an IgG, and / or a combination thereof.

[00328] In various embodiments, use of a mouse as described herein to generate a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence for making a human antigen-binding protein is provided, further comprising fusing the amplified <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain variable region sequence to human heavy or light chain constant region sequences or optionally a human heavy chain variable region seugence, expressing the fused sequences in a cell, and recovering the expressed sequences thereby generating a human antigen-binding protein.

[00329] In various embodiments, the human heavy chain constant regions are selected from IgM, IgD, IgA, IgE and IgG. In various specific embodiments, the IgG is selected from an IgG1, an IgG2, an IgG3 and an IgG4. In various embodiments, the human heavy chain constant region comprises a C<com:Sub>H< / com:Sub>1, a hinge, a C<com:Sub>H< / com:Sub>2, a C<com:Sub>H< / com:Sub>3, a C<com:Sub>H< / com:Sub>4, or a combination thereof. In various embodiments, the light chain constant region is an immunoglobulin <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> constant region. In various embodiments, the cell is selected from a HeLa cell, a DU145 cell, a Lncap cell, a MCF-7 cell, a MDA-MB-438 cell, a PC3 cell, a T47D cell, a THP-1 cell, a U87 cell, a SHSY5Y (human neuroblastoma) cell, a Saos-2 cell, a Vero cell, a CHO cell, a GH3 cell, a PC12 cell, a human retinal cell (e.g., a PER.C6<com:Sup>TM< / com:Sup> cell), and a MC3T3 cell. In a specific embodiment, the cell is a CHO cell.

[00330] In one aspect, a method for generating a human light chain variable region specific against an antigen of interest is provided, comprising the steps of immunizing a mouse as described herein with the antigen, isolating at least one cell from the mouse producing a human light chain variable region specific against the antigen, generating at least one cell producing a human antigen-binding protein comprising the light chain variable region specific against the antigen, and culturing at least one cell producing the human antigen-binding protein, and obtaining said human antigen-binding protein. In one embodiment, the human light chain variable region is a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> region.

[00331] In various embodiments, the at least one cell isolated from the mouse producing a human light chain variable region specific against the antigen is a splenocyte or a B cell.

[00332] In various embodiments, the antigen-binding protein is an antibody.

[00333] In various embodiments, immunization with the antigen of interest is carried out with protein, DNA, a combination of DNA and protein, or cells expressing the antigen.

[00334] In one aspect, use of a mouse as described herein to make a nucleic acid sequence encoding an immunoglobulin variable region or fragment thereof is provided. In one embodiment, the nucleic acid sequence is used to make a human antibody or antigen- binding fragment thereof. In one embodiment, the mouse is used to make an antigen- binding protein selected from an antibody, a multispecific antibody (e.g., a bispecific antibody), an scFv, a bis-scFv, a diabody, a triabody, a tetrabody, a V-NAR, a <com:Math><semantics>VHH<annotation encoding="application / x-tex">V_{HH}< / annotation>< / semantics>< / com:Math>, a <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math>, a <com:Math><semantics>F(ab)<annotation encoding="application / x-tex">F(ab)< / annotation>< / semantics>< / com:Math>, a <com:Math><semantics>F(ab)2<annotation encoding="application / x-tex">F(ab)_2< / annotation>< / semantics>< / com:Math>, a DVD (i.e., dual variable domain antigen-binding protein), a an SVD (i.e., single variable domain antigen-binding protein), or a bispecific T-cell engager (BiTE).

[00335] In one aspect, use of the mouse as described herein for the manufacture of a medicament (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, an antigen-binding protein), or for the manufacture of a sequence encoding a variable sequence of a medicament (e.g., an antigen-binding protein), for the treatment of a human disease or disorder is provided.

[00336] In one aspect, use of a mouse as described herein to make a nucleic acid sequence encoding a first human light chain immunoglobulin variable sequence (V<com:Sub>L< / com:Sub>1) that is cognate with a second human light chain immunoglobulin variable sequence (V<com:Sub>L< / com:Sub>2), wherein the V<com:Sub>L< / com:Sub>1 fused with a human immunoglobulin light chain constant region (polypeptide 1) expresses with V<com:Sub>L< / com:Sub>2 fused with a human immunoglobulin heavy chain constant region (polypeptide 2), as a dimer of polypeptide 1 / polypeptide 2, to form a <com:Math><semantics>VL1−VL2<annotation encoding="application / x-tex">V_L 1 - V_L 2< / annotation>< / semantics>< / com:Math> antibody.

[00337] In one aspect, use of a mouse as described herein to make a nucleic acid sequence encoding a human immunoglobulin light chain variable sequence that is fused with a human immunoglobulin heavy chain sequence, wherein the nucleic acid sequence encodes a human V<com:Sub>L< / com:Sub>-C<com:Sub>H< / com:Sub> polypeptide, wherein the human V<com:Sub>L< / com:Sub>-C<com:Sub>H< / com:Sub> polypeptide expresses as a dimer, and wherein the dimer expresses in the absence of an immunoglobulin light chain (e.g., in the absence of a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> or human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain). In one embodiment, the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math>-<com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math> dimer specifically binds an antigen of interest in the absence of a <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> light chain and in the absence of a <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain.

[00338] In one aspect, use of a mouse as described herein to make a nucleic acid sequence encoding all or a portion of an immunoglobulin variable domain. In one embodiment, the immunoglobulin variable domain is a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> domain.

[00339] In one aspect, use of a nucleic acid construct as described herein for the manufacture of a mouse, a cell, or a therapeutic protein (e.g., an antibody or other antigen- binding protein) is provided.

[00340] In one aspect, use of a nucleic acid sequence from a mouse as described herein to make a cell line for the manufacture of a human therapeutic is provided. In one embodiment, the human therapeutic is a binding protein comprising a human light chain variable sequence (e.g., derived from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segment) fused with a human heavy chain constant sequence. In one embodiment, the human therapeutic comprises a first polypeptide that is a human <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> immunoglobulin light chain, and a second polypeptide that comprises a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> or human <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> variable sequence fused with a human heavy chain constant sequence.

[00341] In one aspect, an expression system is provided, comprising a mammalian cell transfected with a DNA construct that encodes a polypeptide that comprises a somatically mutated human V<com:Sub>L< / com:Sub> domain fused with a human C<com:Sub>H< / com:Sub> domain.

[00342] In one embodiment, the expression system further comprises a nucleotide sequence that encodes an immunoglobulin V<com:Sub>L< / com:Sub> domain fused with a human C<com:Sub>L< / com:Sub> domain, wherein the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain fused with the human <com:Math><semantics>CL<annotation encoding="application / x-tex">C_L< / annotation>< / semantics>< / com:Math> domain is a cognate light chain with the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain fused with the human C<com:Sub>H< / com:Sub> domain.

[00343] In one embodiment, the mammalian cell is selected from a CHO cell, a COS cell, a Vero cell, a 293 cell, and a retinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

[00344] In one aspect, a method for making a binding protein is provided, comprising obtaining a nucleotide sequence encoding a V<com:Sub>L< / com:Sub> domain from a gene encoding a V<com:Sub>L< / com:Sub> region fused to a C<com:Sub>H< / com:Sub> region from a cell of a mouse as described herein, and cloning the nucleotide sequence encoding the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> region sequence in frame with a gene encoding a human <com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math> region to form a human binding protein sequence, expressing the human binding protein sequence in a suitable cell.

[00345] In one embodiment, the mouse has been immunized with an antigen of interest, and the <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> region fused to the <com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math> region specifically binds (e.g., with a <com:Math><semantics>KD<annotation encoding="application / x-tex">K_D< / annotation>< / semantics>< / com:Math> in the micromolar, nanomolar, or picomolar range) an epitope of the antigen of interest. In one embodiment, nucleotide sequence encoding the V<com:Sub>L< / com:Sub> region fused to the C<com:Sub>H< / com:Sub> region is somatically mutated in the mouse.

[00346] In one embodiment, the suitable cell is selected from a B cell, a hybridoma, a quadroma, a CHO cell, a COS cell, a 293 cell, a HeLa cell, and a human retinal cell expressing a viral nucleic acid sequence (e.g., a PERC.6™ cell).

[00347] In one embodiment, the C<com:Sub>H< / com:Sub> region comprises a human IgG isotype. In a specific embodiment, the human IgG is selected from an IgG1, IgG2, and IgG4. In another specific embodiment, the human IgG is IgG1. In another specific embodiment, the human IgG is IgG4. In another specific embodiment, the human IgG4 is a modified IgG4. In one embodiment, the modified IgG4 comprises a substitution in the hinge region. In a specific embodiment, the modified IgG4 comprises a substitution at amino acid residue 228 relative to a wild-type human IgG4, numbered according to the EU numbering index of Kabat. In a specific embodiment, the substitution at amino acid residue 228 is a S228P substitution, numbered according to the EU numbering index of Kabat.

[00348] In one embodiment, the cell further comprises a nucleotide sequence encoding a V<com:Sub>L< / com:Sub> domain from a light chain that is cognate to the V<com:Sub>L< / com:Sub> domain fused to the C<com:Sub>H< / com:Sub> region, and the method further comprises expressing the nucleotide sequence encoding the cognate V<com:Sub>L< / com:Sub> domain fused to a human Cκ or Cλ domain.

[00349] In one aspect, a method for making a bispecific antigen-binding protein is provided, comprising exposing a first mouse as described herein to a first antigen of interest and identifying a sequence of a first human V<com:Sub>L< / com:Sub> domain that specifically binds the first antigen of interest; exposing a second mouse as described herein to a second antigen of interest and identifying a sequence of a second human V<com:Sub>L< / com:Sub> domain that specifically binds the second antigen of interest; wherein the first human V<com:Sub>L< / com:Sub> domain does not bind the second antigen of interest, and the second human V<com:Sub>L< / com:Sub> domain does not bind the first antigen of interest; and fusing the sequence of the first human V<com:Sub>L< / com:Sub> domain to a first heavy chain constant sequence to form a first antigen-binding polypeptide, and fusing the sequence of the second human V<com:Sub>L< / com:Sub> domain to a second heavy chain constant sequence to form a second antigen-binding polypeptide; and, employing the first and the second antigen-binding polypeptides in a bispecific binding protein.

[00350] In one embodiment, the antigen-binding protein further comprises a first immunoglobulin light chain that comprises a human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> variable domain that is cognate with the first human V<com:Sub>L< / com:Sub> domain, and a second immunoglobulin light chain that comprises a human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> or <com:Math><semantics>λ<annotation encoding="application / x-tex">\lambda< / annotation>< / semantics>< / com:Math> variable domain that is cognate with the second human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain.

[00351] In one embodiment, the first heavy chain constant sequence is identical to the second heavy chain constant sequence. In one embodiment, the first heavy chain constant sequence comprises a modification that reduces or eliminates binding of the first heavy chain constant region to protein A, and the second heavy chain constant sequence binds protein A.

[00352] In one embodiment, the first and second human V<com:Sub>L< / com:Sub> domains comprise a sequence derived from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the first and second human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domains comprise a sequence derived from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_\lambda< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the first and second human <com:Math><semantics>V1<annotation encoding="application / x-tex">V_1< / annotation>< / semantics>< / com:Math> domains comprise a sequence derived from a human D<com:Sub>H< / com:Sub> gene segment. In one embodiment, the first and second human V<com:Sub>L< / com:Sub> domains comprise a sequence derived from a human J<com:Sub>H< / com:Sub> gene segment. In a specific embodiment, the first and second human V<com:Sub>L< / com:Sub> domains comprise a sequence derived from a human D<com:Sub>H< / com:Sub> and a human J<com:Sub>H< / com:Sub> gene segment. In a specific embodiment, the first and second human V<com:Sub>L< / com:Sub> domains comprise a sequence derived from a human <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment.

[00353] In one embodiment, the first human V<com:Sub>L< / com:Sub> domain comprises a sequence derived from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment and the second human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain comprises a sequence derived from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segment. In one embodiment, the first human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain comprises a sequence derived from a human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V_{\lambda}< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segment and the second human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> domain comprises a sequence derived from a human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and a human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segment.

[00354] In one aspect, an immunoglobulin variable region (VR) (<com:Math><semantics>e.g.<annotation encoding="application / x-tex">e.g.< / annotation>< / semantics>< / com:Math>, comprising a human <com:Math><semantics>VL<annotation encoding="application / x-tex">V_L< / annotation>< / semantics>< / com:Math> sequence fused with a human <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math>, or <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math>, or <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math>, or <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JL<annotation encoding="application / x-tex">J_L< / annotation>< / semantics>< / com:Math>) made in a mouse as described herein is provided. In a specific embodiment, the immunoglobulin VR is derived from a germline human gene segment selected from a <com:Math><semantics>Vκ<annotation encoding="application / x-tex">V\kappa< / annotation>< / semantics>< / com:Math> segment and a <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> segment, wherein the VR is encoded by a rearranged sequence from the mouse wherein the rearranged sequence is somatically hypermutated. In one embodiment, the rearranged sequence comprises 1 to 5 somatic hypermutations. In one embodiment, the rearranged sequence comprises at least 6, 7, 8, 9, or 10 somatic hypermutations. In one embodiment, the rearranged sequence comprises more than 10 somatic hypermutations. In one embodiment, the rearranged sequence is fused with one or more human or mouse heavy chain constant region sequences (e.g., selected from a human or mouse <com:Math><semantics>CH1<annotation encoding="application / x-tex">C_{H1}< / annotation>< / semantics>< / com:Math>, hinge, <com:Math><semantics>CH2<annotation encoding="application / x-tex">C_{H2}< / annotation>< / semantics>< / com:Math>, C<com:Sub>H< / com:Sub>3, and a combination thereof).

[00355] In one aspect, an immunoglobulin variable domain amino acid sequence of a binding protein made in a mouse as described herein is provided. In one embodiment, the VR is fused with one or more human or mouse heavy chain constant region sequences (e.g., selected from a human or mouse C<com:Sub>H< / com:Sub>1, hinge, C<com:Sub>H< / com:Sub>2, C<com:Sub>H< / com:Sub>3, and a combination thereof).

[00356] In one aspect, a light chain variable domain encoded by a nucleic acid sequence derived from a mouse as described herein is provided.

[00357] In one aspect, an antibody or antigen-binding fragment thereof (e.g., Fab, F(ab)<com:Sub>2< / com:Sub>, scFv) made in a mouse as described herein, or derived from a sequence made in a mouse as described herein, is provided. BRIEF DESCRIPTION OF THE FIGURES

[00358] FIG. 1 illustrates schematics (not to scale) of the mouse heavy chain locus (top) and the human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain locus (bottom). The mouse heavy chain locus is about 3 Mb in length and contains approximately 200 heavy chain variable (V<com:Sub>H< / com:Sub>) gene segments, 13 heavy chain diversity (D<com:Sub>H< / com:Sub>) gene segments and 4 heavy chain joining (J<com:Sub>H< / com:Sub>) gene segments as well as enhancers (Enh) and heavy chain constant (<com:Math><semantics>CH<annotation encoding="application / x-tex">C_H< / annotation>< / semantics>< / com:Math>) regions. The human <com:Math><semantics>κ<annotation encoding="application / x-tex">\kappa< / annotation>< / semantics>< / com:Math> light chain locus is duplicated into distal and proximal contigs of opposite polarity spanning about 440 kb and 600 kb, respectively. Between the two contigs is about 800 kb of DNA that is believed to be free of <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments. The human <com:Math><semantics>K<annotation encoding="application / x-tex">K< / annotation>< / semantics>< / com:Math> light chain locus contains about 76 <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments, 5 Jκ gene segments, an intronic enhancer (Enh) and a single constant region <com:Math><semantics>(Cκ)<annotation encoding="application / x-tex">(C\kappa)< / annotation>< / semantics>< / com:Math>.

[00359] FIG. 2 shows an exemplary targeting strategy for progressive insertion of 40 human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and five human <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments into the mouse heavy chain locus resulting in a modified mouse immunoglobulin heavy chain locus comprising human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments operably linked to mouse immunoglobulin heavy chain constant regions. Hygromycin (hyg) and neomycin (neo) selection cassettes are shown with recombinase recognition sites (R1, R2, etc.).

[00360] FIG. 3 shows an exemplary targeting strategy for progressive insertion of a plurality of human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> and a single human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segment into the mouse heavy chain locus. Hygromycin (hyg) and neomycin (neo) selection cassettes are shown with recombinase recognition sites (R1, R2, etc.).

[00361] FIG. 4 shows an exemplary targeting strategy for progressive insertion of a plurality of human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math> and four human <com:Math><semantics>Jλ<annotation encoding="application / x-tex">J\lambda< / annotation>< / semantics>< / com:Math> gene segments into the mouse heavy chain locus. Hygromycin (hyg) and neomycin (neo) selection cassettes are shown with recombinase recognition sites (R1, R2, etc.).

[00362] FIG. 5 shows an exemplary targeting strategy for progressive insertion of human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math>, human <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> and human <com:Math><semantics>JH<annotation encoding="application / x-tex">J_H< / annotation>< / semantics>< / com:Math> gene segments into the mouse heavy chain locus. Hygromycin (hyg) and neomycin (neo) selection cassettes are shown with recombinase recognition sites (R1, R2, etc.).

[00363] FIG. 6 shows an exemplary targeting strategy for progressive insertion of human <com:Math><semantics>Vλ<annotation encoding="application / x-tex">V\lambda< / annotation>< / semantics>< / com:Math>, human <com:Math><semantics>DH<annotation encoding="application / x-tex">D_H< / annotation>< / semantics>< / com:Math> and human <com:Math><semantics>Jκ<annotation encoding="application / x-tex">J\kappa< / annotation>< / semantics>< / com:Math> gene segments into the mouse heavy chain locus. Hygromycin (hyg) and neomycin (neo) selection cassettes are shown with recombinase recognition sites (R1, R2, etc.).

[00364] FIG. 7 shows the steps for cloning a genomic fragment encoding mouse ADAM6 genes from a mouse immunoglobulin heavy chain V-D intergenic region and engineering steps to modify the genomic fragment for insertion into a modified immunoglobulin heavy chain locus.

[00365] FIG. 8 shows a targeting strategy for insertion of a genomic fragment encoding mouse ADAM6 genes into the Vκ-Jκ intergenic region of a modified mouse immunoglobulin heavy chain locus containing human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> and <com:Math><semantics>JK<annotation encoding="application / x-tex">J_K< / annotation>< / semantics>< / com:Math> gene segments operably linked to mouse immunoglobulin heavy chain constant regions.

[00366] FIG. 9 shows a targeting strategy for insertion of a genomic fragment encoding mouse ADAM6 genes upstream (5') of human <com:Math><semantics>VK<annotation encoding="application / x-tex">V_K< / annotation>< / semantics>< / com:Math> gene segments (i.e., <com:Math><semantics>hVK2<annotation encoding="application / x-tex">hV_K2< / annotation>< / semantics>< / com:Math>-40) of a modified mouse immunoglobulin heavy chain locus containing human Vκ and Jκ gene segments operably linked to mouse immunoglobulin heavy chain constant regions. DETAILED DESCRIPTION

[00367] This invention is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention is defined by the claims.

[00368] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, particular methods and materials are now described.

[00369] The phrase "substantial" or "substantially" when used to refer to an amount of gene segments (e.g., "substantially all" V gene segments) includes both functional and non- functional gene segments and include, in various embodiments, e.g., 80% or more, 85% or more, 90% or more, 95% or more 96% or more, 97% or more, 98% or more, or 99% or more of all gene segments; in various embodiments, "substantially all" gene segments includes, e.g., at least 95%, 96%, 97%, 98%, or 99% of functional (i.e., non-pseudogene) gene segments.

[00370] The term "replacement" includes wherein a DNA sequence is placed into a genome of a cell in such a way as to replace a sequence within the genome with a heterologous sequence (e.g., a human sequence in a mouse), at the locus of the genomic sequence. The DNA sequence so placed may include one or more regulatory sequences that are part of source DNA used to obtain the sequence so placed (e.g., promoters, enhancers, 5'- or 3'-untranslated regions, appropriate recombination signal sequences, etc.). For example, in various embodiments, the replacement is a substitution of an endogenous sequence for a heterologous sequence that results in the production of a gene product from the DNA sequence so placed (comprising the heterologous sequence), but ...

Claims

1. A method of making a genetically modified mouse, the method comprising genetically modifying a mouse so that its genome comprises: (a) an insertion of one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (b) an insertion of one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (c) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the ectopic nucleic acid when the genetically modified mouse is male, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse, wherein the genetically modified mouse expresses an antibody comprising (i) a polypeptide that comprises a human light chain variable domain linked to a mouse heavy chain constant domain; and (ii) a polypeptide that comprises a human light chain variable domain linked to a mouse light chain constant domain.

2. The method of claim 1, wherein the mouse immunoglobulin light chain constant region gene is a mouse Cκ.

3. The method of claim 1 or 2, wherein the one or more human V L gene segments and one or more human J L gene segments upstream of the mouse immunoglobulin light chain constant region gene are one or more human V K V_K gene segments and one or more human J K J_K gene segments.

4. The method of any one of claims 1-3, wherein the one or more human V L gene segments and one or more human J L gene segments upstream of the mouse immunoglobulin heavy chain constant region gene are one or more human V κ V_{\kappa} gene segments and one or more human J κ J_{\kappa} gene segments.

5. The method of any one of claims 1-4, wherein the ectopic nucleic acid is present between two of the inserted one or more human V L gene segments and one or more human J L gene segments of (b).

6. The method of claim 5, wherein the inserted one or more human V L gene segments and one or more human J L J_L gene segments are human κ \kappa light chain gene segments or human λ \lambda light chain gene segments.

7. The method of any one of claims 1-6, wherein the inserted one or more human V L gene segments includes V κ 5 − 2 V\kappa 5-2 and V κ 4 − 1 V\kappa 4-1 and the ectopic nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between V κ 5 − 2 V\kappa5-2 and V κ 4 − 1 V\kappa4-1 .

8. The method of any one of claims 1-6, wherein the inserted one or more human V L gene segments includes Vκ4-1 and the inserted one or more human J L gene segments include Jκ1 and the ectopic nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between V κ 4 − 1 V\kappa 4-1 and J κ 1 J\kappa 1 .

9. The method of any one of claims 1-8, wherein the genetically modified mouse comprises one or more endogenous V L and / or one or more endogenous J L gene segments that are incapable of rearranging to form an immunoglobulin light chain variable region in the mouse.

10. The method of any one of claims 1-9, wherein the genetically modified mouse comprises deletion of, or replacement of, one or more endogenous immunoglobulin heavy chain gene sequences.

11. The method of any one of claims 1-10, wherein the ectopic nucleic acid sequence encodes a mouse ADAM6a protein or a functional fragment thereof and a mouse ADAM6b protein or a functional fragment thereof.

12. A mouse B cell derived from the genetically modified mouse made according to the method of any one of claims 1-10, wherein the mouse B cell genome comprises: (a) a rearranged human light chain variable region operably linked to the mouse immunoglobulin light chain constant region gene, wherein the rearranged human light chain variable region includes a human V L gene segment joined with a human J L gene segment; (b) a rearranged human light chain variable region operably linked to the mouse immunoglobulin heavy chain constant region gene, wherein the rearranged human light chain variable region includes a human V L gene segment joined with a human J L gene segment; and (c) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse, wherein the mouse B cell expresses an antibody comprising (i) a polypeptide that comprises a human light chain variable domain linked to a mouse heavy chain constant domain; and (ii) a polypeptide that comprises a human light chain variable domain linked to a mouse light chain constant domain.

13. A mouse cell derived from the genetically modified mouse made according to the method of any one of claims 1-10, wherein the mouse cell genome comprises: (a) an insertion of one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (b) an insertion of one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (c) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse, wherein the cell is an embryonic stem cell, or a pluripotent cell.

14. A hybridoma made from the mouse B cell of claim 12.

15. A method for making an antibody that binds to an antigen of interest, wherein the method comprises (a) exposing a genetically modified mouse made according to the method of any one of claims 1-11 to an antigen of interest; (b) isolating one or more B cells of the mouse, wherein the one or more B cells express an antibody that binds the antigen of interest, and wherein the antibody comprises a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin light chain constant domain and a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin heavy chain constant domain; (c) identifying a nucleic acid that encodes one of the human light chain variable domains of the antibody that binds that antigen of interest; and (d) recombinantly linking the nucleic acid of (c) with a human immunoglobulin constant region nucleic acid to make a human antibody that binds the antigen of interest.

16. A method for making a human light chain variable domain, wherein the method comprises (a) exposing a genetically modified mouse made according to the method of any one of claims 1-11 to an antigen of interest; (b) isolating one or more B cells of the genetically modified mouse, wherein the one or more B cells express an antibody that binds the antigen of interest, and wherein the antibody comprises a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin light chain constant domain and a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin heavy chain constant domain; and (c) identifying one of the human light chain variable domains of an antibody expressed by the one or more B cells of step (b).

17. The method of claim 16, wherein identifying one of the human light chain variable domains of an antibody expressed by the one or more B cells of step (b) comprises identifying a nucleic acid that encodes one of the human light chain variable domains of an antibody expressed by the one or more B cells of step (b).

18. A method for making a human immunoglobulin light chain variable region, wherein the method comprises (a) exposing a genetically modified mouse made according to the method of any one of claims 1-11 to an antigen of interest; (b) isolating one or more B cells of the genetically modified mouse, wherein the one or more B cells express an antibody that binds the antigen of interest, and wherein the antibody comprises a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin light chain constant domain and a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin heavy chain constant domain; and (c) identifying a human light chain variable region nucleic acid that encodes one of the human immunoglobulin light chain variable domains of an antibody expressed by the one or more B cells of step (b).

19. A method for modifying an immunoglobulin heavy chain locus of a mouse, comprising: (a) making a first modification of a mouse immunoglobulin heavy chain locus that results in a reduction or elimination of endogenous ADAM6 activity in a male mouse, wherein the first modification includes insertion of one or more human V L gene segments, one or more human J L gene segments, or a combination thereof; and (b) making a second modification to insert a nucleic acid in an ectopic location in the genome of the mouse that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the nucleic acid when the mouse is male, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse.

20. The method according to claim 19, wherein the first modification further comprises the insertion of one or more human D H gene segments that are capable of rearranging with one or more V L V_L and one or more J L J_L gene segments.

21. The method according to claim 19 or claim 20, wherein the ectopic nucleic acid sequence encodes a mouse ADAM6a protein or a functional fragment thereof and a mouse ADAM6b protein or a functional fragment thereof.

22. A method for generating a human immunoglobulin light chain variable domain comprising the steps of: (a) immunizing a genetically modified mouse with an antigen, wherein the genetically modified mouse: (i) has a germline genome that comprises: (A) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (B) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (C) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the ectopic nucleic acid when the genetically modified mouse is male, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse; (ii) generates antibodies when immunized with the antigen, wherein the antibodies each comprise a human light chain variable domain operably linked to a mouse heavy chain constant domain and a human light chain variable domain operably linked to a mouse light chain constant domain; and (iii) is fertile; and (b) determining a sequence of a human light chain variable domain of an antibody that specifically binds the antigen and that was generated by the genetically modified mouse.

23. The method of claim 22, wherein determining the sequence of a human light chain variable domain comprises determining a nucleic acid sequence that encodes the human light chain variable domain.

24. A method for making a human antibody that binds to an antigen of interest, wherein the method comprises: (a) exposing a genetically modified mouse to an antigen of interest wherein the genetically modified mouse has a germline genome that comprises: (A) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (B) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (C) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the ectopic nucleic acid when the mouse is male, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse; (b) isolating one or more B cells of the genetically modified mouse, wherein the one or more B cells express an antibody that binds the antigen of interest, and wherein the antibody comprises a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin light chain constant domain and a human immunoglobulin light chain variable domain linked to a mouse immunoglobulin heavy chain constant domain; (c) identifying a nucleic acid that encodes one of the human light chain variable domains of the antibody that binds that antigen of interest; and (d) expressing in a mammalian cell in vitro a human antibody comprising a human light chain variable domain encoded by the nucleic acid of (c) operably linked to a human immunoglobulin constant region nucleic acid to make the human antibody that binds the antigen of interest.

25. A method for generating a human immunoglobulin light chain variable region comprising the steps of: (a) immunizing a genetically modified mouse with an antigen, wherein the genetically modified mouse: (i) has a germline genome that comprises: (A) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (B) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (C) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the ectopic nucleic acid when the genetically modified mouse is male, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse; (ii) generates antibodies when immunized with the antigen, wherein the antibodies each comprise a human light chain variable domain operably linked to a mouse heavy chain constant domain and a human light chain variable domain operably linked to a light chain constant domain; and (iii) is fertile; (b) allowing the genetically modified mouse to mount an immune response to the antigen; (C) isolating a B cell from the genetically modified mouse that expresses an antibody that specifically binds the antigen; and (d) amplifying a human light chain variable region that encodes a human light chain variable domain of an antibody that specifically binds the antigen and that was expressed by the isolated B cell of the genetically modified mouse.

26. A method of making a nucleic acid encoding a fully human immunoglobulin light chain comprising the steps of: (a) immunizing a genetically modified mouse with an antigen, wherein the genetically modified mouse: (i) has a germline genome that comprises: (A) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (B) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (C) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the ectopic nucleic acid when the genetically modified mouse is male, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse; (ii) generates antibodies when immunized with the antigen, wherein the antibodies each comprise a human light chain variable domain operably linked to a mouse heavy chain constant domain and a human light chain variable domain operably linked to a mouse light chain constant domain; and (iii) is fertile; (b) allowing the genetically modified mouse to mount an immune response to the antigen; (c) isolating a B cell from the genetically modified mouse that expresses an antibody that specifically binds the antigen; (d) amplifying a human light chain variable region that encodes a light chain variable domain of an antibody that specifically binds the antigen and that was generated by the genetically modified mouse; and (e) operably linking the human light chain variable region to a human heavy or light chain constant region to form a nucleic acid encoding a fully human immunoglobulin chain.

27. A method for making a human antibody, comprising: culturing a host cell comprising a first nucleic acid including a first human light chain variable region operably linked to a human heavy chain constant region and a second nucleic acid including a second human light chain variable region operably linked to a human light chain constant region, so that a human antibody is expressed from the first and second nucleic acids, wherein the first and second human light chain variable regions are each a rearranged V L / J L and were identified from a cell of a genetically modified mouse that includes in its genome: (a) an insertion of one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (b) an insertion of one or more human V L V_L gene segments and one or more human J L J_L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (c) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the ectopic nucleic acid when the mouse is male, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse.

28. The method of any one of claims 22 to 27, wherein the ectopic nucleic acid sequence encodes a mouse ADAM6a protein or a functional fragment thereof and a mouse ADAM6b protein or a functional fragment thereof.

29. The method according to any one of claims 19 to 21, wherein the first and the second modification are made in a single embryonic stem (ES) cell, and the single ES cell is introduced into a host embryo to make the genetically modified mouse.

30. The method of any one of claims 22 to 29, wherein the mouse light chain constant region gene is a mouse Ck gene.

31. The method of any one of claims 22 to 29, wherein the mouse light chain constant region gene is a mouse C λ C\lambda gene.

32. The method of any one of claims 22 to 31, wherein the one or more human V L gene segments and one or more human J L gene segments operably linked to the light chain constant region gene are one or more human V κ V_{\kappa} gene segments and one or more human J κ J_{\kappa} gene segments or one or more human V λ V\lambda gene segments and one or more human J λ J\lambda gene segments.

33. The method of any one of claims 22 to 31, wherein the one or more human V L gene segments and one or more human J L gene segments operably linked to the mouse heavy chain constant region gene are one or more human V K V_K gene segments and one or more human J K J_K gene segments or one or more human V λ V\lambda gene segments and one or more human J λ J\lambda gene segments.

34. The method of any one of claims 22 to 33, wherein: (i) the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between one of the one or more human V L gene segments and one of the one or more human J L gene segments operably linked to the mouse heavy chain constant region gene; (ii) the germline genome of the mouse includes two or more human V L gene segments operably linked to the mouse immunoglobulin heavy chain constant region gene at an endogenous mouse immunoglobulin heavy chain locus and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between two of the two or more human V L gene segments operably linked to the mouse heavy chain constant region gene at an endogenous mouse immunoglobulin heavy chain locus; or (iii) the germline genome of the mouse includes two or more human J L gene segments operably linked to the mouse heavy chain constant region gene at an endogenous mouse immunoglobulin heavy chain locus and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between two of the two or more human J L gene segments operably linked to the mouse heavy chain constant region at an endogenous mouse immunoglobulin heavy chain locus.

35. The method of any one of claims 19-34, wherein the one or more human V L gene segments includes Vκ5-2 and Vκ4-1 and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between V κ 5 V\kappa 5 -2 and V κ 4 V\kappa 4 -1.

36. The method of any one of claims 19-34, wherein the one or more human V L gene segments includes V κ 4 V\kappa 4 -1 and the inserted one or more J L J_L gene segments include J κ 1 J\kappa 1 and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between V κ 4 − 1 V\kappa 4-1 and J κ 1 J\kappa 1 .

37. The method of any one of claims 19-36, wherein the genetically modified mouse comprises one or more endogenous V L and / or one or more endogenous J L gene segments that are incapable of rearranging to form an immunoglobulin light chain variable region in the mouse.

38. The method of any one of claims 19-36, wherein the genetically modified mouse comprises deletion of, or replacement of, one or more endogenous immunoglobulin heavy chain genes.

39. An embryonic stem (ES) cell whose genome comprises: (a) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin light chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin light chain constant region gene; (b) one or more human V L gene segments and one or more human J L gene segments upstream of a mouse immunoglobulin heavy chain constant region gene, wherein the one or more human V L gene segments and one or more human J L gene segments are operably linked to the mouse immunoglobulin heavy chain constant region gene; and (c) an ectopic nucleic acid that encodes a mouse ADAM6a protein or a functional fragment thereof and / or a mouse ADAM6b protein or a functional fragment thereof, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof are expressed from the ectopic nucleic acid, wherein the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof improve or restore fertility when expressed in a male mouse, wherein the cell is not a fertilized egg or a totipotent cell.

40. The ES cell of claim 39, wherein the mouse immunoglobulin light chain constant region gene is a mouse C κ C\kappa gene.

41. The ES cell of claim 39, wherein the mouse immunoglobulin light chain constant region gene is a mouse C λ C\lambda gene.

42. The ES cell of any one of claims 39 to 41, wherein the one or more human V L gene segments and one or more human J L gene segments operably linked to the mouse immunoglobulin light chain constant region gene are one or more human Vκ gene segments and one or more human J κ \kappa gene segments or one or more human V λ \lambda gene segments and one or more human J λ J\lambda gene segments.

43. The ES cell of any one of claims 39 to 42, wherein the one or more human V L gene segments and one or more human J L gene segments operably linked to the mouse immunoglobulin heavy chain constant region gene are one or more human Vκ gene segments and one or more human J κ \kappa gene segments or one or more human V λ \lambda gene segments and one or more human J λ J\lambda gene segments.

44. The ES cell of any one of claims 39 to 43, wherein: (i) the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between one of the one or more human V L gene segments and one of the one or more human J L gene segments operably linked to the mouse immunoglobulin heavy chain constant region gene; (ii) the germline genome of the mouse includes two or more human V L gene segments operably linked to the mouse immunoglobulin heavy chain constant region gene at an endogenous mouse immunoglobulin heavy chain locus and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between two of the two or more human V L gene segments operably linked to the mouse immunoglobulin heavy chain constant region gene at an endogenous mouse immunoglobulin heavy chain locus; or (iii) the germline genome of the mouse includes two or more human J L gene segments operably linked to the mouse immunoglobulin heavy chain constant region gene at an endogenous mouse immunoglobulin heavy chain locus and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between two of the two or more human J L gene segments operably linked to the mouse immunoglobulin heavy chain constant region gene at an endogenous mouse immunoglobulin heavy chain locus.

45. The ES cell of any one of claims 39 to 44, wherein the one or more human V L gene segments includes V κ 5 V\kappa 5 -2 and V κ 4 V\kappa 4 -1 and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between V κ 5 − 2 V\kappa 5-2 and V κ 4 − 1 V\kappa 4-1 .

46. The ES cell of any one of claims 39 to 44, wherein the one or more human V L gene segments includes Vκ4-1 and the inserted one or more J L gene segments include Jκ1 and the nucleic acid that encodes the mouse ADAM6a and / or ADAM6b proteins or functional fragments thereof is present between V κ 4 − 1 V\kappa 4-1 and J κ 1 J\kappa 1 .

47. The ES cell of any one of claims 39 to 46, wherein the ES cell is a mouse ES cell, and the mouse comprises one or more endogenous V L and / or one or more endogenous J L gene segments that are incapable of rearranging to form an immunoglobulin light chain variable region in the mouse.

48. The ES cell of any one of claims 39 to 47, wherein the ES cell is a mouse ES cell, and the mouse comprises deletion of, or replacement of, one or more endogenous immunoglobulin heavy chain gene sequences.