Transgenic mammals and methods of use thereof

Abstract

To provide transgenic mammals expressing bovine-based immunoglobulins, including transgenic rodents expressing bovine-based immunoglobulins for the development of bovine therapeutic antibodies.SOLUTION: A transgenic rodent or rodent cell is provided comprising an engineered partially bovine immunoglobulin light chain locus comprising bovine immunoglobulin λ light chain variable region gene segments, wherein the engineered immunoglobulin locus is capable of expressing immunoglobulins comprising bovine variable domains, and wherein the transgenic rodent produces or is likely to produce more immunoglobulins comprising λ light chains than immunoglobulins comprising κ light chains.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 869,415, filed July 1, 2019, the disclosure of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on June 23, 2020, is named 0133-0007WO1_SL.txt, and is 119,740 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to the production of immunoglobulin molecules, including methods for producing transgenic mammals capable of producing bovine antigen-specific antibody-secreting cells for monoclonal antibody production. [Background technology]

[0004] background In the following description, several documents and methods are described for background and introductory purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the documents and methods cited herein do not constitute prior art under applicable legal provisions.

[0005] Antibodies have emerged as important biological medicines because (i) they exhibit exquisite binding specificity that allows them to target antigens in diverse molecular forms, (ii) they are physiological molecules with favorable pharmacokinetics that make them well tolerated in human and animal treatments, and (iii) they are associated with potent immunological properties that naturally combat infectious agents. Furthermore, established techniques exist for rapidly isolating antibodies from laboratory animals that can readily mount specific antibody responses against virtually any foreign substance not naturally present in the body.

[0006] In their most basic form, antibodies consist of two identical heavy (H) chains, each paired with an identical light (L) chain. The N-terminus of both the H and L chains contains a variable domain (V H and V L ), which together provide a heavy-light chain pair with unique antigen-binding specificity.

[0007] Antibody V H and V L The exons encoding the domains are not present in germline DNA. Instead, each V H The exon is a randomly selected V exon present in the immunoglobulin heavy chain locus (IGH). H , D and J H produced by recombination of gene segments; similarly, individual V L The exon is a randomly selected V in the light chain locus. L and J. L It is produced by chromosomal rearrangement of gene segments.

[0008] The bovine genome contains two alleles capable of expressing heavy chains (one from each parent), two alleles capable of expressing kappa (κ) light chains, and two alleles capable of expressing lambda (λ) light chains. H , D and J H There are gene segments, as well as multiple V in both the immunoglobulin kappa (IGK) and immunoglobulin lambda (IGL) light chain loci. L and J. L There are gene segments (Collins and Watson (2018) Immunoglobulin Light Chain Gene Rearrangements, Receptor Editing and the Development of a Self-Tolerant Antibody Repertoire. Front. Immunol. 9:2249. (doi: 10.3389 / fimmu.2018.02249)).

[0009] In a typical immunoglobulin heavy chain variable region locus, V HThe gene segment is J H Located upstream (5') of the gene segment, the D gene segment is V H Gene segments and J H Located between gene segments. J at the IGH locus H Downstream (3') of the gene segment is the antibody constant region (C H ) is a cluster of exons encoding C H Each cluster of exons encodes a different antibody class (isotype). In mice, there are eight classes of antibodies: IgM, IgD, IgG3, IgG1, IgG2a (or IgG2c), IgG2b, IgE, and IgA (referred to at the nucleic acid level as μ, δ, γ3, γ1, γ2a / c, γ2b, ε, and α, respectively). In bovine animals, the putative isotypes are IgM, IgD, IgG, IgE, and IgA (Figure 12A). There are three subtypes of bovine IgG and two IgMs. The functional consequences of having two IgM subtypes are unknown, but ultralong CDR H3 antibodies appear to be exclusively used by IgM2 (Stanfield et al. (2018) The unusual genetics and biochemistry of bovine immunoglobulins Adv. Immunol. 137:136-164 (doi: 10.1016 / bs.ai.2017.12.004)). The bovine IGH locus is small, approximately 680 Kb.

[0010] The question of whether there is bovine IgD is controversial. Previous studies have found no evidence of the Cδ gene or IgD protein in cattle (Butler, et al. (1996)). The swine Ig heavy chain locus has a single J Hand no identifiable IgD. Int. Immunol. 8:1897-1094 (DOI: 10.1093 / intimm / 8.12.1897); Naessens J (1997) Surface Ig on B lymphocytes from cattle and sheep. Int. Immunol. 9:349-354 (DOI:10.1093 / intimm / 9.3.349). Subsequently, evidence for the presence of the bovine Cδ gene h (Zhao, et al. (2002) J. Immunol. Artiodactyl IgD: The missing link. 160:4408-4416 (DOI:10.4049 / jimmunol.169.8.4408) and bovine IgD expression (Xu, et al. (2012) PLoS ONE Expressional Analysis of Immunoglobulin D in Cattle (Bos taurus), a Large Domesticated Ungulate. 7:e44719 (doi.org / 10.1371 / journal.pone.0044719)) was reported, but IgD + The frequency of cells was much lower than in mice. The current annotation of the bovine IGH locus describes five Cδ genes, IGHDD1P, IGHDD2P, IGHDD39, and IGHD, none of which are functional. There are two IGHD alleles, one of which is a pseudogene due to a frameshift in M1, and the other is an ORF due to a non-canonical splice donor in NGC rather than NGT. The IgD observed by Xu et al. + The low frequency of cells is likely due to occasional leaky splicing from the ORF allele.

[0011] In most mammalian species, the IGK locus is V κ Clusters of gene segments are formed by a small number of J κ Located upstream of the gene segment, J κ The gene segment cluster consists of a single C κ This configuration of the K locus is located upstream of the gene (V κ) a ···(J κ ) b ···C κ (where a and b are independently integers greater than or equal to 1.) The bovine IGK locus is small, approximately 400 Kb.

[0012] The IGL loci of most species are J λ Gene segments and C λ V located 5' to a variable number of JC tandem cassettes consisting of gene segments λ The λ locus contains a set of gene segments (see schematic of the bovine IGL locus in Figure 12B). λ ) a ···(J λ -C λ ) b (where a and b are independently an integer greater than or equal to 1). The mouse IGL locus can be represented as λ ) a ···(J λ -C λ ) b The bovine IGL locus is small, approximately 580 Kb.

[0013] During B cell development, gene rearrangement occurs first on one of the two homologous chromosomes containing the heavy chain variable gene segment. H The exon is then C for IgM H chain expression at the RNA level. μ It is spliced ​​into exons. L -J L Rearrangement occurs with one of the L chain alleles until a functional L chain is produced, after which the L chain polypeptide can combine with an IgM H chain homodimer to form a fully functional B cell receptor (BCR) for the antigen. In mice and humans, IgD is co-expressed with IgM as B cells continue to mature, and IgD is expressed at levels 10-fold higher than IgM in the majority of B cell populations.

[0014] In mice and humans, V L -JL The rearrangement occurs first at the IGK locus on both chromosomes and then at the IGL light chain locus on one chromosome. L -J L It is widely recognized by experts in the field that prior events occur before the mouse becomes recombination-competent. This is supported by the fact that in mouse B cells that express kappa light chains, the lambda loci on both chromosomes are commonly inactivated by non-productive rearrangements. This may explain the predominant kappa light chain usage in mice, which is >90% kappa and <10% lambda.

[0015] However, the ratio of kappa to lambda immunoglobulins varies between species. Many livestock and companion animals are highly lambda dominant, with cattle producing approximately 91% lambda chains. Arun et al. (1996) Immunohistochemical examination of light chain expression (lambda / kappa ratio) in canine, feline, equine, bovine, and porcine plasma cells. Zentralblatt fuer Veterinaermedizin Reihe A. 43:573-576.

[0016] Upon encountering an antigen, B cells then μ and C δ Another round of DNA recombination at the IGH locus to remove the exon, C H This effectively switches the domain to one of the downstream isotypes (a process called class switching).

[0017] Although the genes encoding various bovine (e.g., domestic cattle) and mouse immunoglobulins have been characterized, the sequence and annotation of bovine Ig loci in genome databases remains incomplete. For example, Sinclair, et al., J. Immunol., 159(8):3883-89 (1997), characterized the bovine IgG repertoire as a single diverse V Hdescribed as being biased towards gene segment families; Lopez, et al., Immunol. Rev. 162(1):55-66 (1998) found that single V H described the family and long CDR3 as targets for hypermutation in bovine IgG heavy chains; Hosseini, et al., in Int. Immunol. 16(6):355-63 (1998), described the bovine J-protein complexes that contribute to the Ig repertoire. H describes the antigen-binding position in a bovine antibody as an ultralong CDR3 loop, with a stalk that has a protruding knob, each of which may further vary due to the number of Cys residues and changes in the pattern and connectivity of disulfide bonds produced in somatic cells; Wang, et al., Cell, 153(6):1379-1393 (2013).

[0018] The production of transgenic animals, such as mice with multiple immunoglobulin loci, allows for the use of such transgenic animals in a variety of biological systems and in a variety of research and development applications, such as drug discovery and basic research. For example, the production of transgenic mice carrying human immunoglobulin genes is described in International Applications WO90 / 10077 and WO90 / 04036. WO90 / 04036 describes transgenic mice into which human immunoglobulin "mini" loci have been integrated. WO90 / 10077 describes vectors containing immunoglobulin locus activation regions for use in producing transgenic animals.

[0019] For example, numerous methods have been developed to modify endogenous immunoglobulin variable region gene loci in mice with human immunoglobulin sequences to generate partially or fully human antibodies for drug discovery purposes. Examples of such mice are those described in, for example, U.S. Patents 7,145,056; 7,064,244; 7,041,871; 6,673,986; 6,596,541; 6,570,061; 6,162,963; 6,130,364; 6,091,001; 6,023,010; 5,593,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,661,016; 5,612,205; and 5,591,669. However, the majority of fully humanized immunoglobulin transgenic mice exhibit suboptimal antibody production because B cells in these mice are severely hindered by inefficient V(D)J recombination and the inability of fully human antibodies / BCRs to function optimally with mouse signaling proteins. Other humanized immunoglobulin transgenic mice, in which mouse coding sequences are "swapped" with human sequences, are extremely time-consuming and expensive due to the approach of replacing individual mouse exons with their syntenic human counterparts.

[0020] The use of antibodies that function as drugs is not limited to the prevention or treatment of human diseases. The high population densities of modern intensively managed livestock operations result in the sharing of both resident bacterial flora and pathogens, which rapidly spreads infectious agents. As a result, livestock generally require aggressive infection control strategies, often including the use of antibiotics. The extensive use of antibiotics supports the development of resistant microorganisms, posing a risk to both farm animals and the humans who consume them. As such, there is increasing pressure to reduce or eliminate the use of antibiotics in livestock, such as cattle. Summary of the Invention [Problem to be solved by the invention]

[0021] Based on the foregoing, there is a clear need for an efficient and cost-effective method of producing bovine antibodies to treat diseases in domestic cattle. More specifically, there is a need for a small, rapidly breeding mammal capable of producing antigen-specific bovine immunoglobulins that can be used to generate hybridomas capable of large-scale production of bovine monoclonal antibodies. [Means for solving the problem]

[0022] summary This Summary is provided to introduce selected concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the inventive subject matter, nor is it intended to be used to limit the scope of the inventive subject matter. Other features, details, utilities, and advantages of the inventive subject matter will be apparent from the following Detailed Description, including the embodiments illustrated in the accompanying drawings and defined in the appended claims.

[0023] Described herein are non-bovine mammalian cells and non-bovine mammals having genomes comprising exogenously introduced, partially bovine immunoglobulin loci, where the introduced loci comprise coding sequences of bovine immunoglobulin variable region gene segments and non-coding sequences based on the endogenous immunoglobulin variable region loci of the non-bovine mammalian host. Thus, the non-bovine mammalian cells or mammals can express chimeric B cell receptors (BCRs) or antibodies comprising completely bovine heavy and light chain variable regions with respective constant regions native to the non-bovine mammalian host cell or mammal. In some embodiments, the transgenic cells and animals have genomes in which some or all of the endogenous immunoglobulin variable region gene loci have been removed.

[0024] At a minimum, production of chimeric bovine monoclonal antibodies in a non-bovine mammalian host requires that the host have at least one locus that expresses a chimeric bovine immunoglobulin heavy or light chain. In most embodiments, there will be one heavy chain locus and two light chain loci that express chimeric bovine immunoglobulin heavy and light chains, respectively.

[0025] In some embodiments, the partially bovine immunoglobulin locus is a non-bovine mammalian host endogenous V H Located at the gene locus, bovine V H In these embodiments, the partially bovine immunoglobulin locus further comprises endogenous D and J sequences of the non-bovine mammalian host cell genome. H Bovine D and J sequences, along with non-coding regulatory or scaffolding sequences located proximal to the gene segments H Contains gene segment coding sequences.

[0026] In some embodiments, a bovine immunoglobulin locus is embedded in a non-coding regulatory or scaffold sequence present in an endogenous immunoglobulin heavy chain locus of a non-bovine mammalian host. H , D and J H In some embodiments, the partially bovine immunoglobulin locus comprises a bovine V gene segment coding sequence integrated into a non-coding regulatory or scaffold sequence present in the endogenous immunoglobulin heavy chain locus of a rodent, such as a mouse. H , D and J H In other embodiments, the partially bovine immunoglobulin locus comprises an endogenous V gene segment coding sequence of the non-bovine mammalian host. L Located at the gene locus, bovine V L In one embodiment, the bovine V gene comprises a coding sequence and a non-coding regulatory sequence or scaffold sequence. L The exogenously introduced, partially bovine immunoglobulin locus comprising a coding sequence further comprises a bovine L chain J gene segment coding sequence and non-coding regulatory or scaffolding sequences present proximal to an endogenous L chain J gene segment in the genome of the non-bovine mammalian host cell. In some embodiments, the partially bovine immunoglobulin locus comprises a bovine V L chain J gene segment coding sequence integrated into non-coding regulatory or scaffolding sequences present in an immunoglobulin light chain locus of the non-bovine mammalian host. λ and J. λIn some embodiments, the partially bovine immunoglobulin locus comprises a bovine V gene segment coding sequence integrated into a non-coding regulatory or scaffold sequence present in the immunoglobulin locus of a non-bovine mammalian host. κ and J. κ The non-bovine mammalian host comprises a gene segment coding sequence. In some embodiments, the endogenous κ locus of the non-bovine mammalian host is inactivated or replaced with a sequence encoding a bovine λ chain to increase production of bovine λ immunoglobulin light chains over bovine κ chains. In some embodiments, the endogenous κ locus of the non-bovine mammalian host is inactivated rather than replaced with a sequence encoding a bovine λ chain.

[0027] In some embodiments, the non-bovine mammal is a rodent, for example, a mouse or a rat.

[0028] In some embodiments, the engineered immunoglobulin locus comprises a partially bovine immunoglobulin light chain locus comprising one or more bovine λ variable region gene segment coding sequences, hi some embodiments, the engineered immunoglobulin locus is a partially bovine immunoglobulin light chain locus comprising one or more bovine κ variable region gene segment coding sequences.

[0029] In some embodiments, transgenic rodents or rodent cells are provided having a genome comprising an engineered, partially bovine immunoglobulin locus. In some embodiments, transgenic rodents or rodent cells are provided having a genome comprising an engineered, partially bovine immunoglobulin light chain locus. In some embodiments, the partially bovine immunoglobulin light chain locus of the rodent or rodent cell comprises one or more bovine immunoglobulin λ variable region gene segment coding sequences. In some embodiments, the partially bovine immunoglobulin light chain locus of the rodent or rodent cell comprises one or more bovine immunoglobulin κ variable region gene segment coding sequences. In some embodiments, the engineered immunoglobulin locus is capable of expressing immunoglobulins comprising bovine variable domains.

[0030] In some embodiments, transgenic rodents are provided that produce more immunoglobulins comprising lambda light chains than immunoglobulins comprising kappa light chains. In some embodiments, the transgenic rodent produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and up to about 100% lambda light chain immunoglobulins. In some embodiments, the transgenic rodent produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and up to about 100% lambda light chain immunoglobulins comprising bovine variable domains. In some embodiments, more λ light chain producing cells may be isolated from a transgenic rodent than κ light chain producing cells, hi some embodiments, more cells producing λ light chains with bovine variable domains may be isolated from a transgenic rodent than cells producing κ light chains with bovine variable domains.

[0031] In some embodiments, transgenic rodent cells are provided that are more likely to produce immunoglobulins comprising a λ light chain than immunoglobulins comprising a κ light chain. In some embodiments, the rodent cells are isolated from a transgenic rodent described herein. In some embodiments, the rodent cells are recombinantly produced as described herein. In some embodiments, the transgenic rodent cells or their progeny have at least about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% probability of producing λ light chain immunoglobulins, and up to about 100% probability. In certain embodiments, the transgenic rodent cells or their progeny have at least about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% probability and up to about 100% probability of producing lambda light chain immunoglobulins with bovine variable domains.

[0032] In some embodiments, the engineered partially bovine immunoglobulin locus is a bovine Vλ Gene segment coding sequence and J λ Gene segments include coding sequences as well as non-coding sequences such as regulatory or scaffolding sequences of a rodent immunoglobulin light chain variable region gene locus.

[0033] In some embodiments, the engineered immunoglobulin locus comprises a bovine V-type IgG1 gene integrated into a rodent non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin λ light chain variable region gene locus. λ and J. λ In some embodiments, the engineered immunoglobulin locus comprises a bovine V gene segment coding sequence integrated into the non-coding regulatory or scaffold sequence of a rodent immunoglobulin kappa light chain variable region gene locus. λ and J. λ In some embodiments, the partially bovine immunoglobulin locus comprises one or more bovine V gene segments encoding sequences. λ Gene segment coding sequence and J λ It includes gene segment coding sequences as well as one or more rodent immunoglobulin lambda constant region coding sequences.

[0034] In some embodiments, the engineered immunoglobulin variable region locus is selected from one or more bovine V λ a gene segment coding sequence and one or more JC units, wherein each JC unit is a bovine J λ Gene segment coding sequence and rodent region C λ In some embodiments, the engineered immunoglobulin variable region locus comprises one or more bovine V λ a gene segment coding sequence and one or more JC units, wherein each JC unit is a bovine J λ Gene segment coding sequences and rodent C λ In some embodiments, the rodent C λ The coding sequence of the rodent C λ1 , C λ2 or C λ3 In some embodiments, one or more bovine V coding sequences are selected from the group consisting of: λThe gene segment coding sequence is located upstream of one or more JC units, where each JC unit is a bovine J λ Gene segment coding sequences and rodent C λ In some embodiments, one or more bovine V gene segments are included. λ The gene segment coding sequence is located upstream of one or more JC units, where each JC unit is a bovine J λ Gene segment coding sequences and rodent C λ Gene segment coding sequences and rodent C λ In some embodiments, the JC unit comprises a bovine JC sequence integrated into a non-coding regulatory or scaffold sequence of a rodent immunoglobulin κ light chain locus. λ Gene segment coding sequences and rodent C λ Contains the region coding sequence.

[0035] In some embodiments, one or more rodent V κ gene segment coding sequences and one or more rodent J κ The gene segment coding sequence is deleted, each of which contains one or more bovine V λ A gene segment coding sequence and one or more J λ gene segment coding sequence and at the locus rodent C κ The coding sequence is rodent C λ1 , C λ2 or C λ3 Transgenic rodents or rodent cells are provided that have engineered immunoglobulin loci, including a rodent immunoglobulin κ locus, replaced with coding sequences.

[0036] In some embodiments, the engineered immunoglobulin locus is one or more rodent C λ One or more bovine J genes upstream of the coding sequence λ Upstream of and in the same transcriptional direction as the gene segment coding sequence, one or more bovine V λ Contains gene segment coding sequences.

[0037] In some embodiments, the engineered immunoglobulin locus is one or more rodent C λ One or more bovine J genes upstream of the coding sequence λ one or more bovine V genes in the upstream and reverse transcriptional direction of the gene segment coding sequence λ Contains gene segment coding sequences.

[0038] In some embodiments, the endogenous rodent immunoglobulin kappa light chain locus is deleted, inactivated or a. Total endogenous rodent V κ deletions or mutations in gene segment coding sequences; b. All endogenous rodent J κ deletions or mutations in gene segment coding sequences; c. Endogenous rodent C κ Deletions or mutations in coding sequences; d.J κ Gene segments and C κ Deletions or mutations of splice donor sites, pyrimidine tracts, or splice acceptor sites within introns between exons; and e. Endogenous intron kappa enhancer (iE κ ), 3' enhancer sequence (3'E κ ) or a combination of deletions, mutations, or disruptions The present invention provides a transgenic rodent or rodent cell that is rendered non-functional by one or more of the following:

[0039] In some embodiments, the endogenous rodent immunoglobulin lambda light chain variable domain is a. Total endogenous rodent V λ deletions or mutations of gene segments; b. All endogenous rodent J λ deletions or mutations of gene segments; c. All endogenous rodent C λ Deletions or mutations in the coding sequence; and d.J λ Gene segments and C λDeletion or mutation of a splice donor site, pyrimidine tract, or splice acceptor site, or a combination thereof, within an intron between exons Transgenic rodents or rodent cells are provided in which the gene encoding the nucleotide sequence of the present invention is repressed or inactivated by one or more of the following:

[0040] In some embodiments, a transgenic rodent or rodent cell is provided in which an engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine variable domain and a rodent constant domain. In some embodiments, a transgenic rodent or rodent cell is provided in which an engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine λ variable domain and a rodent λ constant domain. In some embodiments, a transgenic rodent or rodent cell is provided in which an engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine κ variable domain and a rodent κ constant domain.

[0041] In some embodiments, the genome of the transgenic rodent or rodent cell is a bovine V κ and J. κ Transgenic rodents or rodent cells are provided that contain engineered immunoglobulin loci comprising gene segment coding sequences. In one embodiment, a bovine V κ and J. κ The gene segment coding sequence is inserted into a rodent immunoglobulin kappa light chain locus. κ and J. κ The gene segment coding sequence is integrated into a non-coding regulatory or scaffold sequence of a rodent immunoglobulin kappa light chain variable region gene locus. κ and J. κ The coding sequence is inserted upstream of a rodent immunoglobulin kappa light chain constant region coding sequence.

[0042] In some embodiments, the genome of the transgenic rodent or rodent cell contains a bovine V gene inserted into an engineered immunoglobulin locus containing a rodent immunoglobulin λ light chain locus. κand J. κ A transgenic rodent or rodent cell is provided that comprises a gene segment coding sequence. In one embodiment, a bovine V κ and J. κ The gene segment coding sequence is integrated into rodent non-coding regulatory or scaffolding sequences of a rodent immunoglobulin lambda light chain variable region gene locus. In some embodiments, the genome of the transgenic rodent or rodent cell is integrated into a bovine V κ and J. κ The gene segment coding sequence includes a rodent immunoglobulin κ light chain constant region coding sequence inserted downstream of the gene segment coding sequence. In some embodiments, the rodent immunoglobulin κ light chain constant region is inserted upstream of an endogenous rodent Cλ coding sequence. In some embodiments, the rodent immunoglobulin κ light chain constant region is inserted upstream of an endogenous rodent Cλ coding sequence. λ2 In some embodiments, expression of an endogenous rodent immunoglobulin lambda light chain variable domain is achieved by inserting the endogenous rodent immunoglobulin lambda light chain variable domain upstream of the coding sequence. a. Total endogenous rodent V λ deletions or mutations in gene segment coding sequences; b. All endogenous rodent J λ deletions or mutations in gene segment coding sequences; c. Total endogenous C λ Deletions or mutations in the coding sequence; and d.J λ Gene segments and C λ Deletion or mutation of a splice donor site, pyrimidine tract, or splice acceptor site within an intron between exons is inhibited or inactivated by one or more of the following:

[0043] In some embodiments, the engineered partial bovine immunoglobulin light chain locus contains a rodent intronic kappa enhancer (iE κ ) and 3'κ enhancer (3'E κ ) control sequences.

[0044] In some embodiments, the transgenic rodent or rodent cell further comprises an engineered, partially bovine immunoglobulin heavy chain locus comprising bovine immunoglobulin heavy chain variable region gene segment coding sequences and non-coding regulatory and scaffolding sequences of a rodent immunoglobulin heavy chain locus. In some embodiments, the engineered bovine immunoglobulin heavy chain locus comprises a bovine V H , D and J H In one embodiment, each bovine / rodent chimeric V H , D or J H The gene segment was integrated into the non-coding regulatory and scaffolding sequences of the rodent immunoglobulin heavy chain locus, V H , D or J H In some embodiments, the heavy chain scaffold sequence is interspersed with one or both of the functional ADAM6 genes.

[0045] In some embodiments, rodent regulatory and scaffold sequences include one or more enhancers, promoters, splice sites, introns, recombination signal sequences, or combinations thereof.

[0046] In some embodiments, the endogenous rodent immunoglobulin locus of the transgenic rodent or rodent cell is inactivated, hi some embodiments, the endogenous rodent immunoglobulin locus of the transgenic rodent or rodent cell is deleted and replaced with an engineered partial bovine immunoglobulin locus.

[0047] In some embodiments, the rodent is a mouse or a rat. In some embodiments, the rodent cell is an embryonic stem (ES) cell or an early embryonic cell. In some embodiments, the rodent cell is a mouse or rat embryonic stem (ES) cell or an early embryonic mouse or rat cell.

[0048] In some embodiments, cells of the B lymphocyte lineage obtained from a transgenic rodent described herein are provided, wherein the B cells express or are capable of expressing a chimeric immunoglobulin heavy or light chain comprising a bovine variable region and a rodent immunoglobulin constant region. In some embodiments, hybridoma cells or immortalized cell lines are provided that are derived from cells of the B lymphocyte lineage obtained from a transgenic rodent or rodent cell described herein.

[0049] In some embodiments, an antibody or antigen-binding portion thereof produced by a cell from a transgenic rodent or rodent cell described herein is provided.

[0050] In some embodiments, the V is derived from an immunoglobulin produced by a transgenic rodent or rodent cell described herein. H , D or J H or V L Or J L In one embodiment, a method for producing a non-bovine mammalian cell comprising, in part, a bovine immunoglobulin locus is provided, comprising: a) introducing two or more recombinase target sites into the genome of the non-bovine mammalian host cell, integrating at least one site upstream and at least one site downstream of a genomic region comprising an endogenous immunoglobulin variable region gene, wherein the endogenous immunoglobulin variable region gene is a V H , D and J H Gene segment or V κ and J. κ Gene segment or V λ and J. λ Gene segment or V λ , J λ and C λand b) introducing into a non-bovine mammalian host cell by recombinase-mediated cassette exchange (RMCE) an engineered partially bovine immunoglobulin variable gene locus comprising bovine immunoglobulin variable region gene coding sequences as well as non-coding regulatory sequences or scaffold sequences that correspond to non-coding regulatory sequences or scaffold sequences present at an endogenous immunoglobulin variable region gene locus of the non-bovine mammalian host.

[0051] In other embodiments, the method includes, prior to step b, deleting the genomic region flanked by two exogenously introduced recombinase target sites.

[0052] In a specific embodiment, bovine V H gene segment coding sequences and further comprising i) bovine D and J H An exogenously introduced, engineered, partially bovine immunoglobulin heavy chain locus is provided, comprising a gene segment coding sequence and ii) a non-coding regulatory sequence or scaffold sequence upstream of the bovine D gene segment (pre-D sequence, FIG. 1A) that corresponds to a sequence present upstream of the endogenous D gene segment in the genome of the non-bovine mammalian host. In some embodiments, these upstream scaffold sequences are interspersed with non-immunoglobulin genes, such as ADAM6A or ADAM6B (FIG. 1A), which are required for male fertility (Nishimura et al. Developmental Biol. 233(1): 204-213 (2011)). The partially bovine immunoglobulin heavy chain locus is located on the same chromosome as the endogenous immunoglobulin V gene segment coding sequence. H Upstream of the gene locus and endogenous J H It is introduced into the host cell using a recombinase target site that has been previously introduced downstream of the gene locus.

[0053] In other embodiments, the non-coding regulatory sequence or scaffold sequence is derived (at least in part) from another source, e.g., a rationally designed artificial sequence or an otherwise conserved sequence of unknown function, a combination of bovine and artificial or other designed sequences, or sequences from other species. As used herein, "artificial sequence" refers to a nucleic acid sequence that is not derived from a sequence that naturally occurs at a gene locus. In some embodiments, the non-coding regulatory sequence or scaffold sequence is derived from a non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin heavy chain variable region locus. In some embodiments, the non-coding regulatory sequence or scaffold sequence has at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to a non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin heavy chain variable region locus. In other embodiments, the non-coding regulatory sequence or scaffold sequence is a rodent immunoglobulin heavy chain variable region non-coding or scaffold sequence.

[0054] In yet another embodiment of the method, the introduced engineered partially bovine immunoglobulin locus is bovine immunoglobulin V. L In some embodiments, the engineered partially bovine immunoglobulin locus comprises an endogenous immunoglobulin V gene segment coding sequence, and further comprises i) a bovine L chain J gene segment coding sequence and ii) a non-coding regulatory sequence or scaffold sequence that corresponds to the non-coding regulatory sequence or scaffold sequence present at the endogenous L chain locus of the non-bovine mammalian host cell genome. In some embodiments, the engineered partially bovine immunoglobulin locus is a non-coding regulatory sequence or scaffold sequence that corresponds to the endogenous immunoglobulin V gene segment coding sequence of the same chromosome. L It is introduced into the host cell using recombinase target sites previously introduced upstream of the gene locus and downstream of the endogenous J gene locus.

[0055] In a more specific embodiment, bovine V λ Gene segment coding sequences and bovine J λ An exogenously introduced, engineered, partially bovine immunoglobulin light chain locus is provided, comprising a gene segment coding sequence. In some embodiments, the partially bovine immunoglobulin light chain locus is a locus that encodes an endogenous immunoglobulin V locus on the same chromosome. λ Upstream of the gene locus and endogenous J λIt is introduced into the host cell using a recombinase target site that has been previously introduced downstream of the gene locus.

[0056] In some embodiments, the exogenously introduced, engineered, partially bovine immunoglobulin light chain locus is a bovine V κ Gene segment coding sequences and bovine J κ In some embodiments, the bovine immunoglobulin light chain locus is a gene segment coding for an endogenous immunoglobulin V locus on the same chromosome. κ Upstream of the gene locus and endogenous J κ It is introduced into the host cell using a recombinase target site that has been previously introduced downstream of the gene locus.

[0057] In some embodiments, the non-coding regulatory sequence or scaffold sequence is derived from a non-coding regulatory sequence or scaffold sequence of a rodent lambda immunoglobulin light chain variable region locus. In some embodiments, the non-coding regulatory sequence or scaffold sequence has at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to a non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin lambda light chain variable region locus. In other embodiments, the non-coding regulatory sequence or scaffold sequence is a rodent immunoglobulin lambda light chain variable region non-coding or scaffold sequence.

[0058] In some embodiments, the non-coding regulatory sequence or scaffold sequence is derived from a non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin kappa light chain variable region locus. In some embodiments, the non-coding regulatory sequence or scaffold sequence has at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to a non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin kappa light chain variable region locus. In other embodiments, the non-coding regulatory sequence or scaffold sequence is a rodent immunoglobulin kappa light chain variable region non-coding or scaffold sequence.

[0059] In some embodiments, the engineered partially bovine immunoglobulin locus is synthesized as a single nucleic acid and introduced into the non-bovine mammalian host cell as a single nucleic acid region. In some embodiments, the engineered partially bovine immunoglobulin locus is synthesized as two or more contiguous segments and introduced into the mammalian host cell as separate segments. In other embodiments, the engineered partially bovine immunoglobulin locus is produced using recombinant methods and isolated prior to introduction into the non-bovine mammalian host cell.

[0060] In another embodiment, there is provided a method for producing a non-bovine mammalian cell comprising an engineered partially bovine immunoglobulin locus, comprising: a) introducing into the genome of a non-bovine mammalian host cell two or more sequence-specific recombination sites that cannot recombine with each other, wherein at least one recombination site is introduced upstream of an endogenous immunoglobulin variable region gene locus and at least one recombination site is introduced downstream of an endogenous immunoglobulin variable region gene locus on the same chromosome; and b ... on the same chromosome; providing a vector comprising an engineered partial bovine immunoglobulin locus, wherein the partial bovine immunoglobulin locus is flanked by the same two sequence-specific recombination loci that are flanked by an endogenous immunoglobulin variable region gene locus of the host cell of a); c) introducing into the host cell the vector of step b) and a site-specific recombinase that can recognize the two recombinase loci; and d) allowing a recombination event to occur between the genome of the cell of a) and the engineered partial bovine immunoglobulin locus, resulting in replacement of the endogenous immunoglobulin variable region gene locus with the engineered partial bovine immunoglobulin variable region gene locus.

[0061] In some embodiments, the partially bovine immunoglobulin locus is V H Immunoglobulin gene segment coding sequences, further comprising: i) bovine D and J Hgene segment coding sequences, ii) individual Vs endogenously present in the genome of the non-bovine mammalian host H , D and J H and iii) a pre-D sequence based on the endogenous genome of the non-bovine mammalian host cell. The recombinase target site is an endogenous immunoglobulin V H Upstream and endogenous D and J loci H It is introduced downstream of the gene locus.

[0062] In one embodiment, a transgenic rodent is provided having a genome in which a rodent endogenous immunoglobulin variable gene locus has been deleted, wherein the deleted rodent endogenous immunoglobulin variable gene locus has been replaced with an engineered partial bovine immunoglobulin locus comprising bovine immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the rodent endogenous immunoglobulin variable gene locus, wherein the engineered partial bovine immunoglobulin locus of the transgenic rodent is functional and expresses immunoglobulin chains having bovine variable domains and rodent constant domains. In one embodiment, the engineered partial bovine immunoglobulin locus comprises a bovine V H , D and J H In some embodiments, the engineered partially bovine immunoglobulin locus comprises a bovine V L and J. L In some embodiments, the partially bovine immunoglobulin locus comprises a bovine V λ and J. λ In other embodiments, the partially bovine immunoglobulin locus comprises a bovine V κ and J. κ Contains coding sequences.

[0063] Certain embodiments provide cells of a B lymphocyte lineage from a transgenic rodent, part or all of an immunoglobulin molecule comprising a bovine variable domain and a rodent constant domain obtained from a cell of a B lymphocyte lineage, a hybridoma cell derived from a cell of a B lymphocyte lineage, part or all of an immunoglobulin molecule comprising a bovine variable domain and a rodent constant domain obtained from a hybridoma cell, part or all of an immunoglobulin molecule comprising a bovine variable domain obtained from an immunoglobulin molecule obtained from a hybridoma cell, an immortalized cell derived from a cell of a B lymphocyte lineage, part or all of an immunoglobulin molecule comprising a bovine variable domain and a rodent constant domain obtained from an immortalized cell, part or all of an immunoglobulin molecule comprising a bovine variable domain obtained from an immunoglobulin molecule obtained from an immortalized cell.

[0064] In some embodiments, the transgenic rodent comprises an engineered partially bovine immunoglobulin locus, such as a bovine V L and J. L coding sequences and transgenic rodents, wherein the engineered partially bovine immunoglobulin locus is a bovine V H , D and J H or V L and J. L A transgenic rodent is provided, comprising a coding sequence. In one embodiment, the rodent is a mouse. In one embodiment, the non-coding regulatory sequence comprises the following sequences of endogenous host origin: a promoter, introns, splice sites, and a recombination signal sequence for V(D)J recombination before each V gene segment coding sequence; in other embodiments, the engineered partial bovine immunoglobulin locus further comprises one or more of the following sequences of endogenous host origin: an ADAM6A or ADAM6B gene, a Pax-5-activating intergenic repeat (PAIR) element, or a CTCF binding site from heavy chain intergenic regulatory region 1.

[0065] In some embodiments, the non-bovine mammalian cells for use in each of the above methods are mammalian cells, such as mammalian embryonic stem (ES) cells. In some embodiments, the mammalian cells are cells of an early embryo. In some embodiments, the non-bovine mammalian cells are rodent cells. In some embodiments, the non-bovine mammalian cells are mouse cells.

[0066] Once the cells have been subjected to replacement of the endogenous immunoglobulin variable region loci with the introduced partial bovine immunoglobulin variable region loci, the cells can be selected and isolated. In one embodiment, the cells are non-bovine mammalian ES cells, e.g., rodent ES cells, and at least one isolated ES cell clone is then isolated and utilized to generate a transgenic non-bovine mammal expressing the engineered partial bovine immunoglobulin variable region loci.

[0067] In one embodiment, a method of producing a transgenic rodent comprises: a) introducing into the genome of a rodent cell at least one target site for a site-specific recombinase upstream of an endogenous immunoglobulin variable gene locus and at least one target site for a site-specific recombinase downstream of the endogenous immunoglobulin variable gene locus, wherein the endogenous immunoglobulin variable locus is a V H , D and J H Gene segment or V κ and J. κ Gene segment or V λ and J. λ Gene segment or V λ , J λ and C λb) a vector comprising an engineered partial bovine immunoglobulin locus, the engineered partial bovine immunoglobulin locus comprising chimeric bovine immunoglobulin gene segments, wherein each of the partial bovine immunoglobulin gene segments comprises bovine immunoglobulin variable gene coding sequences and rodent non-coding regulatory or scaffold sequences, the partial bovine immunoglobulin variable gene locus flanked by target sites for a site-specific recombinase, wherein the target sites are capable of recombining with target sites introduced into a rodent cell; and (d) allowing a recombination event to occur between the genome of the cell and the engineered, partially bovine immunoglobulin locus, resulting in replacement of the endogenous immunoglobulin variable gene locus with the engineered, partially bovine immunoglobulin locus; and (e) selecting the cells containing the engineered, partially bovine immunoglobulin variable locus produced in step (d); and using the cells to create a transgenic rodent containing the partially bovine engineered, partially bovine immunoglobulin variable locus. In some embodiments, the cells are rodent embryonic stem (ES) cells, and in some embodiments, the cells are mouse embryonic stem (ES) cells. Some embodiments of this method further include, after step (a) and before step (b), deleting the endogenous immunoglobulin variable gene locus by introducing a recombinase that recognizes a first set of target sites, wherein the deleting step leaves in its place at least one set of target sites that cannot recombine with the genome of the rodent cell. In one embodiment, the vector is a bovine V H , D and J H , coding sequence, and in some embodiments, the vector is a bovine V L and J. L In some embodiments, the vector further comprises a rodent promoter, introns, splice sites, and recombination signal sequences for the variable region gene segments.

[0068] In another embodiment, there is provided a method for producing a transgenic non-bovine mammal comprising an exogenously introduced, engineered, partially bovine immunoglobulin variable region gene locus, comprising: a) introducing into the genome of a non-bovine mammalian host cell one or more sequence-specific recombination sites that are flanked by endogenous immunoglobulin variable region gene loci and are unable to recombine with each other; and b) providing a vector comprising the partially bovine immunoglobulin locus having i) bovine variable region gene coding sequences and ii) non-coding regulatory sequences or scaffold sequences based on the endogenous host immunoglobulin variable region gene loci, wherein the coding sequences and non-coding regulatory sequences or scaffold sequences are transfected into the host cell of a). the cells are flanked by the same sequence-specific recombination loci as those introduced into the genome of the cells; c) introducing into the cells the vector of step b) and a site-specific recombinase capable of recognizing the set of recombinase loci; d) allowing recombination events to occur between the genome of the cells of a) and the engineered partial bovine immunoglobulin variable region gene loci, resulting in replacement of the endogenous immunoglobulin variable region gene loci with the partial bovine immunoglobulin loci; e) selecting the cells containing the partial bovine immunoglobulin loci; and f) using the cells to produce a transgenic animal comprising the partial bovine immunoglobulin loci.

[0069] In a specific embodiment, the engineered partially bovine immunoglobulin locus is a bovine V immunoglobulin locus present in the endogenous genome of a non-bovine mammalian host. H , D and J H The gene segment comprises coding sequences and non-coding regulatory and scaffolding pre-D sequences (including fertility genes). In some embodiments, the sequence-specific recombination site then aligns with the endogenous immunoglobulin V H Upstream gene segments and endogenous J H It is introduced downstream of the gene segment.

[0070] In one embodiment, a method for producing a transgenic non-bovine animal comprising an engineered, partially bovine immunoglobulin locus comprises: a) providing a non-bovine mammalian cell having a genome comprising two sets of sequence-specific recombination sites that cannot recombine with each other and are flanked by endogenous immunoglobulin variable region gene loci of the host genome; b) deleting portions of the endogenous immunoglobulin loci of the host genome by introducing a recombinase that recognizes the first set of sequence-specific recombination sites, wherein such deletion in the genome retains the second set of sequence-specific recombination sites; and c) producing an engineered, partially bovine immunoglobulin variable region comprising bovine coding sequences and non-coding regulatory or scaffold sequences based on the endogenous immunoglobulin variable region gene loci. providing a vector comprising the gene locus, wherein the coding and non-coding regulatory sequences or scaffold sequences are flanked by a second set of sequence-specific recombination sites; d) introducing into the cell a site-specific recombinase capable of recognizing the vector of step c) and the second set of sequence-specific recombination sites; e) allowing a recombination event to occur between the genome of the cell and the partial bovine immunoglobulin locus, resulting in replacement of the endogenous immunoglobulin locus with the engineered partial bovine immunoglobulin variable locus; f) selecting cells comprising the partial bovine immunoglobulin variable region gene locus; and g) utilizing the cells to produce a transgenic animal comprising the engineered partial bovine immunoglobulin variable region gene locus.

[0071] In one embodiment, a method for producing a transgenic non-bovine mammal comprising an engineered partial bovine immunoglobulin locus is provided, comprising: a) providing a non-bovine mammalian embryonic stem ES cell having a genome comprising two sequence-specific recombination sites that cannot recombine with each other and are flanked by endogenous immunoglobulin variable region gene loci; and b) providing a vector comprising the engineered partial bovine immunoglobulin locus comprising bovine immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the endogenous immunoglobulin variable region gene loci, wherein the partial bovine immunoglobulin locus is capable of recombining with the endogenous immunoglobulins in the ES cell. the ES cells and vector are flanked by the same two sequence-specific recombination sites flanking the engineered partial bovine immunoglobulin variable region gene loci; c) contacting the ES cells and vector with a site-specific recombinase capable of recombinase recognizing the two recombinase sites under conditions suitable to promote recombination events resulting in replacement of the endogenous immunoglobulin variable region gene loci in the ES cells with the engineered partial bovine immunoglobulin variable region gene loci; d) selecting ES cells containing the engineered partial bovine immunoglobulin loci; and e) using the cells to produce transgenic animals containing the engineered partial bovine immunoglobulin loci.

[0072] In some embodiments, the transgenic non-bovine mammal is a rodent, eg, a mouse or a rat.

[0073] In certain embodiments, non-bovine mammalian cells and non-bovine transgenic mammals are provided that express bovine variable region gene coding sequences and introduced immunoglobulin variable region gene loci with non-coding regulatory or scaffolding sequences based on endogenous non-bovine immunoglobulin loci of the host genome, wherein the non-bovine mammalian cells and transgenic animals express chimeric antibodies with complete bovine heavy or light chain variable domains along with the respective constant regions that are native to the non-bovine mammalian cell or animal.

[0074] Additionally, B cells from transgenic animals capable of expressing partially bovine antibodies with fully bovine variable sequences are provided, where such B cells are immortalized to provide a source of monoclonal antibodies specific for a particular antigen. In one embodiment, cells of the B lymphocyte lineage from transgenic animals are provided that are capable of expressing partially bovine heavy or light chain antibodies comprising bovine variable regions and rodent constant regions.

[0075] In one embodiment, bovine immunoglobulin variable region gene sequences cloned from B cells are provided for use in producing or optimizing antibodies for diagnostic, prophylactic and therapeutic uses.

[0076] In some embodiments, hybridoma cells are provided that are capable of producing partially bovine monoclonal antibodies having complete bovine immunoglobulin variable region sequences. In some embodiments, hybridomas or immortalized cell lines of B lymphocyte lineage are provided.

[0077] In another aspect, an antibody, or antigen-binding portion thereof, produced by the transgenic animal or cell described herein is provided. In another aspect, an antibody, or antigen-binding portion thereof, is provided that comprises a variable heavy chain or variable light chain sequence derived from an antibody produced by the transgenic animal or cell described herein.

[0078] In one embodiment, heavy and light chain immunoglobulin variable domains from monoclonal antibody-producing hybridomas or primary plasma cells or B cells are sequenced and V is generated to produce fully bovine antibodies that are not immunogenic when injected into cattle. H and V L Methods for combining sequences with bovine constant regions are provided.

[0079] These and other aspects, objects and features are described in further detail below. [Brief explanation of the drawings]

[0080] [Figure 1A]Schematic diagram of the endogenous mouse IGH locus located at the telomeric end of chromosome 12.

[0081] [Figure 1B] FIG. 1 is a schematic diagram of the endogenous mouse IGL locus located on chromosome 16.

[0082] [Figure 1C] Schematic diagram of the endogenous mouse IGK locus located on chromosome 6.

[0083] [Figure 2] FIG. 1 is a schematic diagram illustrating a targeting strategy by homologous recombination for introducing a first set of sequence-specific recombination sites into the upstream region of the heavy chain variable region gene locus in the genome of a non-bovine mammalian host cell.

[0084] [Figure 3] FIG. 1 is another schematic diagram illustrating a targeting strategy by homologous recombination for introducing a first set of sequence-specific recombination sites into the upstream region of the heavy chain variable region gene locus in the genome of a non-bovine mammalian host cell.

[0085] [Figure 4] FIG. 1 is a schematic diagram illustrating the introduction of a second set of sequence-specific recombination sites into a region downstream of the heavy chain variable region gene locus in the genome of a non-bovine mammalian cell by a homology targeting vector.

[0086] [Figure 5] FIG. 1 is a schematic diagram illustrating the deletion of an endogenous immunoglobulin heavy chain variable region gene locus from the genome of a non-bovine mammalian host cell.

[0087] [Figure 6] FIG. 1 is a schematic illustrating the RMCE strategy for the introduction of an engineered, partially bovine immunoglobulin heavy chain locus into a non-bovine mammalian host cell genome that has been previously modified to delete the endogenous immunoglobulin heavy chain variable region gene locus.

[0088] [Figure 7] FIG. 1 is a schematic illustrating an RMCE strategy for the introduction of an engineered, partially bovine immunoglobulin heavy chain locus containing additional regulatory sequences into a non-bovine mammalian host cell genome that has been previously modified to delete the endogenous immunoglobulin heavy chain variable region gene.

[0089] [Figure 8] FIG. 1 is a schematic diagram illustrating the introduction of an engineered, partially bovine immunoglobulin heavy chain variable region gene locus into an endogenous immunoglobulin heavy chain locus in the mouse genome.

[0090] [Figure 9] FIG. 1 is a schematic diagram illustrating the introduction of an engineered, partially bovine immunoglobulin κ light chain variable region gene locus into the endogenous immunoglobulin κ light chain locus of the mouse genome.

[0091] [Figure 10] FIG. 1 is a schematic diagram illustrating the introduction of an engineered, partially bovine immunoglobulin λ light chain variable region gene locus into the endogenous immunoglobulin λ light chain locus of the mouse genome.

[0092] [Figure 11] FIG. 1 is a schematic diagram illustrating the introduction of an engineered, partially bovine immunoglobulin locus containing a bovine VH minilocus by RMCE.

[0093] [Figure 12A] Schematic diagram of the endogenous bovine IGH locus (1201) located on chromosome 21. The VH (1202), D (1203), and JH (1204) gene segments show the exons encoding the M1 (1205), DD1P (1206), DD2P (1207), DD3P (1208), M2 (1209), D (1210), G3 (1211), G1 (1212), G2 (1213), E (1214), and A (1215) constant regions. [Figure 12B]Schematic diagram of the endogenous bovine IGL locus (1217) located on chromosome 17. The Vλ (1218) and tandem cluster Jλ-Cλ (1219) gene segments-exons are indicated. Other non-Ig genes interspersed within the locus are zinc finger protein 280B (1220), zinc finger protein 280A (1221), and melanoma-preferentially expressed antigen (1222). VPREB (1223) encodes the Vλ-like element of the preB cell receptor. Genes 1220-1223 are in the opposite transcriptional direction to the Ig genes.

[0094] [Figure 12C] Schematic diagram of the endogenous bovine IGK locus (1224) located on chromosome 11. The Vκ (1225) and Jκ (1226) gene segments are shown as are the exons encoding Cκ (1227). The gene encoding ribose 5-phosphate isomerase A (1228) is located downstream of the locus in the opposite transcriptional direction.

[0095] [Figure 13] FIG. 1 is a schematic diagram illustrating an engineered partial bovine immunoglobulin light chain variable region locus in which one or more bovine Vλ gene segment coding sequences have been inserted into a rodent immunoglobulin κ light chain locus upstream of one or more bovine Jλ gene segment coding sequences which are upstream of one or more rodent Cλ region coding sequences.

[0096] [Figure 14-1] 1 illustrates a schematic diagram in which one or more bovine Vλ gene segment coding sequences are inserted into a rodent immunoglobulin κ light chain locus upstream of an array of Jλ-Cλ tandem cassettes, where Jλ is of bovine origin and Cλ is of murine origin, Cλ1, Cλ2, or Cλ3. [Figure 14-2] Same as above

[0097] [Figure 15]Flow cytometry analysis of cells expressing bovine BLV1H1, a VH domain with an ultralong HCDR3 having a mouse IgM backbone paired with a bovine (b) Vλ (AF023843.1) or Vκ (BC122795) bound to mouse (m) constant regions Cκ, Cλ1, Cλ2, or Cλ3.

[0098] [Figure 16] 1 shows the results of flow cytometry analysis of cells expressing bovine IGHV B4, a VH domain with a normal-length HCDR3 with a mouse IgM backbone paired with a bovine (b) Vλ (AF023843.1) or Vκ (BC122795) bound to the mouse (m) constant regions Cκ, Cλ1, Cλ2, or Cλ3.

[0099] [Figure 17] Figure 17A shows a Western blot of supernatant (1701) and Figure 17B shows a Western blot of cell lysate (1702) from cells expressing bovine BLV1H12, a VH domain with an ultralong HCDR3 having a mouse IgG2a HC backbone paired with bovine (b) Vλ (AF023843.1) or Vκ (BC122795) bound to mouse (m) constant regions Cκ, Cλ1, Cλ2, or Cλ3.

[0100] [Figure 18] Of the Western blots shown in Figures 17A and 17B, Figure 18A shows a loading control using Myc (1802) and Figure 18B shows a loading control using GAPDH (1803).

[0101] [Figure 19] Figure 19A shows a Western blot of supernatant (1901) and Figure 19B shows a Western blot of cell lysate (1902) from cells expressing bovine BLV1H12, a VH domain with an average-length HCDR3 having a mouse IgG2a HC backbone paired with a bovine (b) Vλ (AF023843.1) or Vκ (BC122795) bound to the mouse (m) constant regions Cκ, Cλ1, Cλ2, or Cλ3.

[0102] [Figure 20] Of the Western blots shown in Figures 19A and 19B, Figure 20A shows a loading control using Myc (2001) and Figure 20B shows a loading control using GAPDH (2002).

[0103] [Figure 21] Shown is the expression of intracellular bovine IGHV BLV1H12 (2101) and bovine IGHV BLV5B8 (2102) with mouse IgD backbone and bovine Vλ-mouse Cλ1 (2103), Cλ2 (2104) or Cλ3 (2105).

[0104] [Figure 22] Cell surface expression of the same bovine construct in Figure 21 is shown, stained with the same antibodies as in Figure 21, and the data layout is the same.

[0105] [Figure 23] Figure 2 shows the expression of intracellular bovine IGHV B4 with a mouse IgD backbone and an average length HCDR3 with a bovine Vλ bound to mouse Cλ1 (2302), Cλ2 (2303), or Cλ3 (2304).

[0106] [Figure 24] Cell surface staining shows expression of bovine IGHV B4 with mouse IgD backbone and bovine Vλ bound to mouse Cλ1 (2402), Cλ2 (2403), or Cλ3 (2404), with cell surface staining data in the same arrangement as in Figure 23. DETAILED DESCRIPTION OF THE INVENTION

[0107] definition Terms used herein are intended to have their plain and ordinary meanings as understood by those of ordinary skill in the art. The following definitions are intended to aid in the understanding of the invention, but are not intended to alter or otherwise limit the meaning of such terms unless specifically indicated.

[0108] As used herein, the term "locus" refers to a chromosomal segment or nucleic acid sequence that is endogenously present in a genome or exogenously introduced (or intended to be introduced) into a genome, respectively. For example, an immunoglobulin locus can include some or all of the genes (i.e., V, D, J gene segments and constant region genes) and intervening sequences (i.e., introns, enhancers, etc.) that support the expression of immunoglobulin heavy or light chain polypeptides. Thus, a locus (e.g., an immunoglobulin heavy chain variable region gene locus) can refer to a particular portion of a larger locus (e.g., a V H , D and J H Similarly, an immunoglobulin light chain variable region gene locus can refer to a specific portion of a larger locus (e.g., a V L and J. L (a portion of an immunoglobulin light chain locus comprising a gene segment). As used herein, the term "immunoglobulin variable region gene" refers to a V, D, or J gene segment that encodes a portion of an immunoglobulin heavy or light chain variable domain. As used herein, the term "immunoglobulin variable region gene locus" refers to part or all of a chromosomal segment or nucleic acid strand that comprises a cluster of V, D, or J gene segments, and may include non-coding regulatory or scaffolding sequences.

[0109] As used herein, "gene segment" refers to a nucleic acid sequence that encodes a portion of a heavy or light chain variable domain of an immunoglobulin molecule. A gene segment may include coding and non-coding sequences. The coding sequence of a gene segment is a nucleic acid sequence that can be translated into a polypeptide, such as a leader peptide and the N-terminus of a heavy or light chain variable domain. The coding sequence of a non-gene segment is a sequence adjacent to the coding sequence, which may include a promoter, 5' untranslated sequences, introns interposed by the coding sequence of the leader peptide, recombination signal sequences (RSSs), and splice sites. A gene segment in the immunoglobulin heavy chain (IGH) locus is a V H , D and J HThe light chain variable region gene segments in the immunoglobulin kappa and lambda light loci are V L and J. L In the kappa light chain, V L and J. L The gene segment is V κ and J. κ Similarly, in the lambda light chain, V L and J. L The gene segment is V λ and J. λ They may be referred to as gene segments or as IGLV and IGLJ.

[0110] The heavy chain constant region is C H or IGHC. C encoding IgM, IgD, IgG1-4, IgE, or IgA. H The exons in the region are C, μ , C δ , Cγ 1~4 , C ε Similarly, immunoglobulin kappa or lambda constant regions may be referred to as C or Cα, respectively. κ or C λ and may be referred to as IGKC or IGLC.

[0111] As used herein, "partially bovine" refers to a nucleic acid strand or its expressed protein and RNA products that contains sequences found at a locus in both bovine and non-bovine mammalian hosts. As used herein, "partially bovine" also refers to animals, including nucleic acid sequences from both bovine and non-bovine mammals, such as rodents. In some embodiments, the partially bovine nucleic acid has coding sequences for bovine immunoglobulin heavy or light chain variable region gene segments and sequences based on non-coding regulatory or scaffold sequences from endogenous immunoglobulin loci of the non-bovine mammal.

[0112] The term "based on," when used in reference to an endogenous non-coding regulatory sequence or scaffold sequence from a non-bovine mammalian host cell genome, refers to a non-coding regulatory sequence or scaffold sequence present at a corresponding endogenous locus in the mammalian host cell genome. In certain embodiments, the term "based on" means that the non-coding regulatory sequence or scaffold sequence present in a part-bovine immunoglobulin locus shares a substantially high degree of homology with the non-coding regulatory sequence or scaffold sequence of the endogenous locus of the host mammal. In certain embodiments, the non-coding sequence at a part-bovine immunoglobulin locus shares at least about 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology with the corresponding non-coding sequence found at the endogenous locus of the host mammal. In certain embodiments, the non-coding sequence at a part-bovine immunoglobulin locus is retained from the immunoglobulin locus of the host mammal. In certain embodiments, the bovine coding sequence is incorporated into a non-regulatory or scaffold sequence of the immunoglobulin locus of the host mammal. In some embodiments, the host mammal is a rodent, such as a rat or mouse.

[0113] "Non-coding regulatory sequences" refer to sequences known to be essential for (i) V(D)J recombination, (ii) isotype switching, (iii) proper expression of full-length immunoglobulin heavy or light chains after V(D)J recombination, and (iv) alternative splicing, for example, to produce membrane and secreted forms of immunoglobulin heavy chains. "Non-coding regulatory sequences" may further include the following sequences of endogenous origin: enhancers and locus control elements such as CTCF and PAIR sequences (Proudhon, et al., Adv. Immunol. 128:123-182 (2015)); promoters preceding each endogenous V gene segment; splice sites; introns; and recombination signal sequences flanking each V, D, or J gene segment. In certain embodiments, the "non-coding regulatory sequences" of a partially bovine immunoglobulin locus share at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and up to 100% homology with the corresponding non-coding sequences found in the target endogenous immunoglobulin locus of the non-bovine mammalian host cell.

[0114] "Scaffold sequence" refers to sequences that intervene between gene segments present at an endogenous immunoglobulin locus in a host cell genome. In some embodiments, the scaffold sequence is interspersed with sequences that are essential for expression of a functional non-immunoglobulin gene, e.g., ADAM6A or ADAM6B. In some embodiments, the scaffold sequence is derived (at least in part) from another source - for example, it can be a rationally designed or artificial sequence, a sequence present at an immunoglobulin locus in the bovine genome, a sequence present at an immunoglobulin locus in another species, or a combination thereof. It is understood that the term "non-coding regulatory or scaffold sequence" has an inclusive meaning (i.e., it refers to both non-coding regulatory sequences and scaffold sequences already present at a locus).

[0115] The term "homology targeting vector" refers to a nucleic acid sequence used to modify the endogenous genome of a mammalian host cell by homologous recombination; such a nucleic acid sequence may contain (i) a targeting sequence that has substantial homology to corresponding endogenous sequences adjacent to the locus present in the genome of the non-bovine mammalian host, (ii) at least one sequence-specific recombination site, (iii) non-coding control or scaffolding sequences, and (iv) optionally, one or more selectable marker genes. As such, a homology targeting vector can be used to introduce sequence-specific recombination sites into specific regions of the host cell genome.

[0116] "Site-specific recombination" or "sequence-specific recombination" refers to the process of DNA rearrangement between two compatible recombination sequences (also referred to as "sequence-specific recombination sites" or "site-specific recombination sequences") that involves any of the following three events: a) deletion of preselected nucleic acids flanking the recombination sites; b) inversion of the nucleotide sequence of preselected nucleic acids flanking the recombination sites; and c) reciprocal exchange of nucleic acid sequences flanking the recombination sites that are located on different nucleic acid strands. It is understood that this reciprocal exchange of nucleic acid segments can be utilized as a targeting strategy for introducing exogenous nucleic acid sequences into the genome of a host cell.

[0117] The term "targeting sequence" refers to a sequence that is homologous to a DNA sequence in the genome of a cell adjacent to or adjacent to the region of the immunoglobulin locus to be modified. The adjacent or adjacent sequence can be located within the locus itself or upstream or downstream of the coding sequence in the genome of the host cell. The targeting sequence is inserted into a recombinant DNA vector used for transfection, for example, an ES cell, so that the sequence to be inserted into the host cell genome, such as the sequence of the recombination site, is adjacent to the targeting sequence of the vector.

[0118] As used herein, the term "site-specific targeting vector" refers to a vector containing nucleic acid encoding a sequence-specific recombination locus, an engineered partial bovine locus, and optionally a selectable marker gene for use in modifying an endogenous immunoglobulin locus in a host for use in recombinase-mediated site-specific recombination. The recombination locus of the targeting vector is suitable for site-specific recombination with other corresponding recombination loci that have been inserted (e.g., by a homology targeting vector) into the genomic sequence of the host cell adjacent to the immunoglobulin locus to be modified. Integration of the engineered partial bovine sequence into the recombination locus in the immunoglobulin locus results in replacement of the endogenous locus with the exogenously introduced partial bovine region.

[0119] The term "transgene" is used herein to refer to genetic material that has been or is to be artificially inserted into the genome of a cell, and particularly a cell of a mammalian host animal. As used herein, the term "transgene" refers to a partly bovine nucleic acid, for example, a partly bovine nucleic acid in the form of an engineered expression construct or targeting vector.

[0120] "Transgenic animal" refers to a non-bovine animal, usually a mammal, that has an exogenous nucleic acid sequence present as an extra chromosomal element in some of its cells or stably integrated into its germline DNA (i.e., in most or all of its genomic sequences). In some embodiments, the partially bovine nucleic acid is introduced into the germline of such a transgenic animal by methods well known in the art, for example, by genetic manipulation of the host animal's embryos or embryonic stem cells.

[0121] "Vector" includes plasmids and viruses and any DNA or RNA molecule, whether autonomously replicating or not, that can be used to transform or transfect cells.

[0122] It should be noted that, as used herein and in the appended claims, the singular includes the plural unless the context dictates otherwise. Thus, for example, reference to a "locus" includes reference to one or more loci, reference to a "method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

[0123] As used herein, the term "or" may mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are not exclusive in nature. The terms "include," "including," and "including" are not limiting.

[0124] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methods that might be used in connection with the inventions described herein.

[0125] When a range of values ​​is provided, it is understood that each value between the upper and lower limits of the range, and every other stated or intervening value in the stated range, is encompassed within the scope of the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the scope of the invention, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either of both of those included limits are also encompassed within the invention.

[0126] The implementation of the techniques described herein may employ conventional techniques and explanations of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry, and sequencing technology that are within the skill of those in the art, unless otherwise specified. Such conventional techniques include polymer array synthesis, polynucleotide hybridization and ligation, polymerase chain reaction, and hybridization detection using labels. Specific descriptions of suitable techniques may be found in the examples herein. However, other equivalent conventional techniques may also be used.Such conventional techniques and descriptions are incorporated herein by reference in their entirety for all purposes, including, but not limited to, Green, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach and Veksler, Eds. (2007), PCR Primer: A Laboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: A Molecular Cloning Manual; Mount (2004), Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New York NY; Gait, These techniques can be found in standard laboratory manuals such as "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., W.H. Freeman Pub., New York, NY; and Berg et al. (2002) Biochemistry, 5th Ed., W.H. Freeman Pub., New York, NY.

[0127] Detailed Description In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one skilled in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and methods known to those skilled in the art have not been described in order to avoid obscuring the present invention.

[0128] Described herein is a transgenic rodent or rodent cell having a genome comprising an engineered, partially bovine immunoglobulin heavy or light chain locus. In some embodiments, the partially bovine immunoglobulin heavy chain locus comprises one or more bovine immunoglobulin heavy chain variable region gene segments. In some embodiments, the partially bovine immunoglobulin light chain locus comprises one or more bovine immunoglobulin lambda light chain variable region gene segments. In some embodiments, the partially bovine immunoglobulin light chain locus comprises one or more bovine immunoglobulin kappa light chain variable region gene segments. In some embodiments, the partially bovine immunoglobulin heavy or light chain locus comprises immunoglobulin region gene segments from a cow (Bos Taurus).

[0129] In some embodiments, non-bovine mammalian cells are provided that contain an exogenously introduced, engineered, partially bovine nucleic acid sequence that includes a coding sequence for a bovine variable region and a non-coding regulatory or scaffold sequence present in an immunoglobulin locus of a mammalian host genome, e.g., a mouse genomic non-coding sequence when the host mammal is a mouse. In some embodiments, one or more coding sequences for a bovine variable region gene segment are integrated into non-coding regulatory or scaffold sequences that correspond to those of an immunoglobulin locus of the mammalian host genome. In some embodiments, the coding sequence for a bovine variable region gene segment is integrated into a non-coding regulatory or scaffold sequence of a rodent or mouse immunoglobulin locus.

[0130] In some embodiments, the bovine immunoglobulin locus is synthetic and under the control of endogenous host regulatory elements.H , D or J H or V L or J L In some embodiments, the partial bovine immunoglobulin locus comprises a bovine V gene segment coding sequence integrated with non-coding regulatory or scaffolding sequences corresponding to those of the immunoglobulin locus in the mammalian host genome. H , D or J H or V L or J L Contains gene segment coding sequences.

[0131] Also provided are methods for producing transgenic rodents or rodent ES cells containing exogenously introduced, engineered, partially bovine immunoglobulin loci, wherein the resulting transgenic rodent is capable of producing more λ light chain-containing immunoglobulins than κ light chain-containing immunoglobulins.

[0132] Immunoglobulin loci in mice and cattle In the humoral immune system, a diverse antibody repertoire is produced through combinatorial and junctional diversity at the IGH and IGL chain loci by a process called V(D)J recombination. In expanding B cells, the first recombination event occurs when one D and one J at the heavy chain locus are recombined. H This occurs between gene segments, and the DNA between these two gene segments is deleted. H After recombination, the newly formed DJ H V from the upstream region of the complex H Gene segments are joined and rearranged. H DJ H Forming an exon. Newly produced V H DJ H Exon recombination V H All other sequences between the D and D gene segments are deleted from the genome of each B cell. This rearranged exon is ultimately expressed on the B cell surface as the variable region of the H chain polypeptide, which binds to the L chain polypeptide to form the B cell receptor (BCR).

[0133] The mouse light chain repertoire is thought to be shaped by the sequence of gene rearrangements. The IGK light chain loci on both chromosomes are V-type, whereas the IGL light chain locus on either chromosome is V-type. λ -J λ Before we can accept recombination, we must first κ -J κ If the initial κ rearrangement is unproductive, further rounds of secondary rearrangement can proceed in a process known as receptor editing (Collins and Watson. (2018) Immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire. Front. 9:2249.). The process of sequential rearrangement of the κ chain locus can continue until all possible recombinations are exhausted. Recombination then proceeds at the second κ chromosome. If, after multiple rearrangements, the second chromosome fails to produce a productive rearrangement, rearrangement at the λ locus continues (Collins and Watson (2018) Immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire. Front. Immunol. 9:2249). This preference for light chain rearrangement is thought to result in a light chain repertoire in mice that is >90% kappa and <10% lambda.

[0134] However, immunoglobulins in the bovine immune system are biased toward lambda light chain usage, estimated at at least 90% lambda to <10% kappa (Arun et al. (1996) Immunohistochemical examination of light-chain expression (lambda / kappa ratio) in canine, feline, equine, bovine, and porcine plasma cells. Zentralbl Veterinarmed A. 43(9):573-6).

[0135] Murine and bovine Ig loci are highly complex in the many properties they contain and how the coding regions diversify through V(D)J rearrangement; however, this complexity does not extend to the fundamental details of the structure of each variable region gene segment. V, D, and J gene segments are highly uniform in composition and organization. For example, V gene segments have the following properties, arranged in an essentially invariant sequential form in immunoglobulin loci: a short transcriptional promoter region (<600 bp long), 5' UTRs, and exons encoding most of the antibody chain signal peptides; introns; exons encoding small portions of the antibody chain signal peptides and most of the antibody variable domains, and a 3' recombination signal sequence required for V(D)J rearrangement. Similarly, D gene segments have the following necessary and invariant properties: a 5' recombination signal sequence, a coding region, and a 3' recombination signal sequence. J gene segments have the following necessary and invariant properties: a 5' recombination signal sequence, a coding region, and a 3' splice donor sequence.

[0136] Non-bovine mammalian cells are provided that contain exogenously introduced, engineered, partially bovine nucleic acid sequences, including bovine variable region coding and non-coding sequences (e.g., promoters, 5' and 3' recombination sequences and splice acceptor sites).

[0137] Compared to humans and mice, bovine germline heavy chain V, D, and J segments are scarce. BoV is encoded on chromosome 21. H The V named 1 H Only the IL-1 family is expressed at the cDNA level. Figure 12A provides a schematic diagram of the endogenous bovine IGH locus (1201) and an expanded view of the IGHC region (1202). The bovine immunoglobulin heavy chain variable region locus is V H (1203), D(1204) and J H (1205) gene segments. The bovine genome contains approximately 20 V H Gene segments, 10 D and 4 functional J HIt is believed to contain gene segments.

[0138] The sequence of bovine IGH is shown in Table 1.

[0139] Due to the limited number of germline heavy chain V, D, and J segments, bovine immunoglobulin diversity relies on somatic hypermutation of the ultralong CDR H3 rather than on germline combinatorial diversity (Stanfield et al. (2018) The unusual genetics and biochemistry of bovine immunoglobulins. Adv. Immunol. 137:135-164). Various breeds of cattle are thought to all have unusually long CDR H3s encoded by long bovine D segments. One bovine D (designated D2) contains 149 nucleotides, corresponding to 49 amino acid codons. This long D contributes significantly to the length of the complementarity-determining region 3 (CDR3) of bovine immunoglobulin heavy chains. By comparison, the average heavy chain CDR3 length in mice is approximately 11 amino acids, and in humans it is approximately 15 amino acids. Bovine antibodies are of two types: conventional antibodies, despite their relatively long CDR3 (approximately 25 amino acids), and those with ultralong CDR3s of 40 to 67 residues, averaging approximately 58 amino acids. The extraordinarily long CDR3s have a unique structure, including a supporting stalk with a protruding knob. In conventional antibodies, the CDR loops of both the heavy and light chain variable regions can contribute to antigen binding. However, in ultralong (UL) CDR3 antibodies, the CDR H1, H2, L1, and L2 loops only form the supporting stalk, while the UL CDR H3 loop forms a knob structure that binds to antigen and can vary greatly due to the number of Cys residues and the resulting changes in the pattern and connectivity of disulfide bonds generated in somatic cells. The heavy chains of all UL bovine antibodies analyzed to date have a single V H Gene segment, VHBUL, single D gene segment, D2 and single J H Gene segment, J H Coded by 1.

[0140] Similar to humans and mice, two types of Ig light chains (κ and λ) are expressed in cattle, although the λ to κ ratio differs significantly between these animals. In mice, approximately 96% of the light chains in serum antibodies are of the κ type, whereas in humans the κ type accounts for only 66% of the total population of Ig L chains. In contrast, the L chain repertoire in cattle is biased towards λ chains. Cattle have 20 functional V λ and eight functional V κ UL bovine antibodies have a single V λ , V λ1x Use.

[0141] The bovine κ locus (1240) is located on chromosome 11 and is approximately 280 kb in size. The κ locus contains 22 V κ Gene (1225), 3 J κ gene (1226) and one C κ gene (1227). Figure 12C provides a schematic diagram of the endogenous bovine IGK locus.

[0142] The bovine λ locus (1216) is located on chromosome 17 and is larger than the κ locus. λ -C λ Approximately 63 Vs arranged in tandem clusters λ Genes (1218), 8 J λ Genes and 9 C λ Contains genes (C λ6 is J λ Figure 12B provides a schematic diagram of the endogenous bovine IGL locus.

[0143] The mouse immunoglobulin κ locus is located on chromosome 6. Figure 1B provides a schematic diagram of the endogenous mouse IGK locus. The IGK locus (112) spans 3300 Kbp and consists of five linked (J) κ ) gene segment (114) and one constant (C κ ) gene (115), located upstream of which are over 100 variable V κ The mouse κ locus contains an intronic enhancer (iE κ, 116), which activates κ rearrangement and helps maintain earlier and more efficient κ rearrangement than λ (Inlay et al. (2004) Important Roles for E Protein Binding Sites within the Immunoglobulin κ chain intronic enhancer in activating V κ J κ rearrangement. J. Exp. Med. 200(9):1205-1211). Other enhancers, such as 3' enhancers (3'E κ , 117) is C κ Located 9.1 Kb downstream of exon iE and involved in kappa rearrangement and transcription κ and 3'E κ Mutant mice lacking both genes express V at the κ locus. κ J κ There is no rearrangement (Inlay et al. (2002) Essential roles of the kappa light chain intronic enhancer and 3' enhancer in kappa rearrangement and demethylation. Nature Immunol. 3(5):463-468). However, for example, the insertion of a neomycin resistance gene into iE κ Most of the destruction of V κ J κ Deletion of the Igκ Light Chain Intronic Enhancer / Matrix Attachment Region Impairs but Does Not Abolish V κ J κ Rearrangement).

[0144] The mouse immunoglobulin λ locus is located on chromosome 16. Figure 1C provides a schematic diagram of the endogenous mouse IGL locus (118). The organization of the mouse immunoglobulin λ locus differs from that of the mouse immunoglobulin κ locus. The locus spans 240 kb and contains three functionally variable (V λ ) gene segments (IGLV2, 119; IGLV3, 120 and IGLV1, 123) and λ linkages (J λ ) gene segments and constant (C λ There are two clusters containing three tandem cassettes of gene segments (IGLJ2, 121; IGLC2, 122; IGLJ3, 124; IGLC3, 125; IGLJ1, 126; IGLC1, 127), where V λ The gene segment is located upstream (5') of a variable number of JC tandem cassettes. The locus also contains three transcriptional enhancers (E λ2~4 , 128;E λ , 129;E λ3~1 , 130).

[0145] The bovine nucleic acid sequences described herein comprise a bovine V gene, while the transgenic animals retain regulatory sequences and other elements that may be found within the intervening sequences of the host genome (e.g., rodent) that help promote efficient antibody production and antigen recognition in the host. H or V L This allows for the production of heavy or light chain repertoires containing the region.

[0146] In some embodiments, the synthetic or recombinantly produced partially bovine nucleic acid comprises a bovine coding sequence and an immunoglobulin V coding sequence. H , V λ or V κ The locus is engineered to contain both non-bovine non-coding regulatory or scaffold sequences from the locus, or in some embodiments, a combination thereof.

[0147] In some embodiments, the transgenic rodent or rodent cell expressing an immunoglobulin having a bovine variable region comprises one or more bovine V HThe gene segment coding sequence is located at the V H In other embodiments, transgenic rodents or rodent cells expressing immunoglobulins with bovine variable regions can be produced by inserting one or more bovine V loci into the L The gene segment coding sequence is located at V of the rodent light chain immunoglobulin locus. L It can be produced by insertion into the locus.

[0148] The presence of two light chain loci - κ and λ - is a key feature of rodent V λ One or more bovine V to locate λ or J λ Insertion of gene segment coding sequence, rodent V κ One or more bovine V to locate κ or J κ Insertion of gene segment coding sequence, rodent V κ One or more bovine V to locate λ or J λ Insertion of gene segment coding sequences and rodent V λ One or more bovine V to locate κ or J κ This means that a variety of light chain insertion combinations, including but not limited to the insertion of gene segment coding sequences, are possible for the production of transgenic rodents or rodent cells expressing immunoglobulins with bovine variable regions.

[0149] The selection and development of transgenic rodents or rodent cells that partially express bovine immunoglobulins is complicated by the fact that more than 90% of the light chains produced by mice are kappa and less than 10% are lambda, whereas more than 90% of the light chains produced by cattle are lambda and less than 10% are kappa.

[0150] Because mice produce primarily κLC-containing antibodies, one rational way to increase the production of λLC-containing partially bovine immunoglobulins by transgenic rodents is to transfect one or more bovine V-proteins into the rodent κ locus. λ or J λHowever, as shown in Example 9 below, the bovine V λ Region exon and rodent C κ Coupling of exons in the region results in suboptimal expression of partially bovine immunoglobulins in vitro.

[0151] Transgenic rodents or rodent cells capable of expressing immunoglobulins comprising bovine variable domains are provided, wherein the transgenic rodents produce or are more likely to produce immunoglobulins comprising λ light chains than immunoglobulins comprising κ light chains. Without wishing to be bound by theory, it is believed that transgenic rodents or rodent cells that produce or are more likely to produce immunoglobulins comprising λ light chains will provide a more complete antibody repertoire for therapeutic development.

[0152] Provided herein is a transgenic rodent or rodent cell having a genome comprising a partially bovine immunoglobulin light chain locus. In some embodiments, the partially bovine immunoglobulin light chain locus comprises a bovine immunoglobulin λ light chain variable region gene segment. In some embodiments, the engineered immunoglobulin locus is capable of expressing an immunoglobulin comprising a bovine variable domain. In some embodiments, the engineered immunoglobulin locus is capable of expressing an immunoglobulin comprising a bovine λ variable domain. In some embodiments, the engineered immunoglobulin locus is capable of expressing an immunoglobulin comprising a bovine κ variable domain. In some embodiments, the engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine variable domain and a rodent constant domain. In some embodiments, the engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine λ variable domain and a rodent λ constant domain. In some embodiments, the engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine kappa variable domain and a rodent kappa constant domain.

[0153] In some embodiments, transgenic rodents or rodent cells are more likely to produce or produce immunoglobulins comprising λ light chains than immunoglobulins comprising κ light chains. In some embodiments, transgenic rodents are provided, wherein more λ light chain-producing cells than κ light chain-producing cells are more likely to be isolated from the rodent. In some embodiments, transgenic rodents are provided that produce at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and up to about 100%, immunoglobulins comprising λ light chains. In some embodiments, transgenic rodent cells or their progeny are provided that are more likely to produce immunoglobulins comprising λ light chains than immunoglobulins comprising κ light chains. In some embodiments, the transgenic rodent cells or their progeny have at least about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% probability of producing immunoglobulins comprising a λ light chain, and up to about 100% probability. In some embodiments, transgenic rodents or rodent cells are provided in which endogenous rodent light chain immunoglobulin loci have been deleted and replaced with engineered partial bovine light chain immunoglobulin loci. In some embodiments, the transgenic rodent is a mouse.

[0154] Immunoglobulin light chain locus In some embodiments, a transgenic rodent or rodent cell is provided having a genome comprising a recombinantly produced, partially bovine immunoglobulin variable region locus. In some embodiments, the partially bovine immunoglobulin variable region locus comprises a light chain variable region (V L In some embodiments, the partially bovine immunoglobulin variable region locus is one or more bovine V λ gene segment coding sequence or one or more bovine J λ In some embodiments, the partially bovine immunoglobulin variable region locus comprises one or more bovine V κ gene segment coding sequence or one or more bovine J κIn some embodiments, the partially bovine immunoglobulin variable region locus comprises one or more rodent constant domain genes or coding sequences. In some embodiments, the partially bovine immunoglobulin variable region locus comprises one or more rodent C constant domain genes or coding sequences. λ In some embodiments, the partially bovine immunoglobulin variable region locus is a gene or coding sequence derived from one or more rodent C κ The gene or coding sequence comprises an endogenous rodent light chain immunoglobulin locus that has been inactivated. In some embodiments, the endogenous rodent light chain immunoglobulin locus has been deleted and replaced with an engineered partial bovine light chain immunoglobulin locus.

[0155] In some embodiments, the engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine λ variable domain and a rodent λ constant domain, hi some embodiments, the engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine κ variable domain and a rodent κ constant domain.

[0156] In some embodiments, the engineered partially bovine immunoglobulin variable region loci are derived from V chromosomes from the bovine genome. λ V containing most or all of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least 20, 30, 40, 50, and up to 63 bovine V loci. λ V containing gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from the bovine genome. λ V, which comprises at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence; L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from Bos Taurus. λ V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence LIncludes sitting position.

[0157] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a J nucleotide sequence found in the bovine genome. λ V containing most or all of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least one, two, or three bovine J loci. λ V containing gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a J locus found in the bovine genome. λ V comprising at least about 50%, 75% and up to 100% of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a J locus from Bos Taurus. λ V comprising at least about 50%, 75% and up to 100% of the gene segment coding sequence L Includes sitting position.

[0158] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a V nucleotide sequence from the bovine genome. λ and J. λ V containing most or all of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from the bovine genome. λ and J. λ V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from Bos Taurus. λ and J. λ V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence L Includes sitting position.

[0159] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a V region that is mostly or entirely derived from the bovine genome. κ V containing gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and up to 22 bovine V loci. κ V containing gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from the bovine genome. κ V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from Bos Taurus. κ V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence L Includes sitting position.

[0160] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a J nucleotide sequence found in the bovine genome. κ V containing most or all of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least one, two, or three bovine J loci. κ V containing gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a J locus found in the bovine genome. κ V comprising at least about 50%, 75% and up to 100% of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a J locus from Bos Taurus. κ V comprising at least about 50%, 75% and up to 100% of the gene segment coding sequenceL Includes sitting position.

[0161] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a V nucleotide sequence from the bovine genome. κ and J. κ V containing most or all of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from the bovine genome. κ and J. κ V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence L In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from Bos Taurus. κ and J. κ V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence L Includes sitting position.

[0162] In some embodiments, the engineered immunoglobulin locus is a bovine V L The engineered immunoglobulin locus comprises a gene segment coding sequence and a rodent non-coding regulatory or scaffold sequence from a rodent immunoglobulin light chain variable region gene locus. In some embodiments, the engineered immunoglobulin locus is a bovine V λ or J λ The engineered immunoglobulin locus comprises a gene segment coding sequence and a rodent non-coding regulatory sequence or scaffold sequence from a rodent immunoglobulin light chain variable region gene locus. In some embodiments, the rodent non-coding regulatory sequence or scaffold sequence is from a rodent immunoglobulin λ light chain variable region gene locus. In some embodiments, the rodent non-coding regulatory sequence or scaffold sequence is from a rodent immunoglobulin κ light chain variable region locus. In some embodiments, the engineered immunoglobulin locus is from a bovine V λ and J. λThe gene segment coding sequence includes a rodent non-coding regulatory sequence or scaffold sequence from a rodent immunoglobulin lambda light chain variable region gene locus. In some embodiments, the partially bovine immunoglobulin locus includes one or more rodent immunoglobulin lambda constant regions (C λ In some embodiments, the partially bovine immunoglobulin locus comprises one or more bovine V λ and J. λ Gene segment coding sequences and one or more rodent immunoglobulin C λ In some embodiments, the engineered immunoglobulin locus comprises a bovine V coding sequence integrated into a rodent non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin λ light chain variable region gene locus. λ and J. λ gene segment coding sequences and one or more rodent C λ Contains coding sequences.

[0163] In some embodiments, the engineered immunoglobulin locus is a bovine V λ or J λ The engineered immunoglobulin locus comprises a bovine V gene segment coding sequence and a rodent non-coding regulatory sequence or scaffold sequence from a rodent immunoglobulin kappa light chain variable region gene locus. In some embodiments, the engineered immunoglobulin locus comprises a bovine V gene segment coding sequence and a rodent non-coding regulatory sequence or scaffold sequence from a rodent immunoglobulin kappa light chain variable region gene locus. λ or J λ In some embodiments, the engineered immunoglobulin locus comprises a bovine V λ and J. λ Gene segment coding sequences and one or more rodent immunoglobulin C λ The engineered immunoglobulin locus comprises a coding sequence and a rodent non-coding regulatory sequence or scaffold sequence from a rodent immunoglobulin kappa light chain variable region gene locus. In some embodiments, the engineered immunoglobulin locus comprises a bovine V coding sequence integrated into a rodent non-coding regulatory sequence or scaffold sequence from a rodent immunoglobulin kappa light chain variable region gene locus. λ and J. λ Gene segment coding sequences and one or more rodent immunoglobulin C λContains coding sequences.

[0164] In one embodiment, one or more bovine V λ The gene segment coding sequence may be derived from one or more rodent C λ One or more Js located upstream of the gene λ Located upstream of the gene segment coding sequence. In some embodiments, one or more bovine V λ The gene segment coding sequence may be one or more rodent lambda C λ One or more Js located upstream of the gene λ It is located upstream of and in the same transcriptional direction as the gene segment coding sequence.

[0165] In some embodiments, the engineered immunoglobulin variable region locus is selected from one or more bovine V λ Gene segment coding sequence, one or more bovine J λ gene segment coding sequences and one or more rodent C λ In some embodiments, the engineered immunoglobulin variable region locus comprises one or more bovine V λ Gene segment coding sequence, one or more bovine J λ gene segment coding sequences and one or more rodent C λ region gene, where V λ and J. λ Gene segment coding sequences and rodent C λ In some embodiments, the engineered immunoglobulin variable region locus is inserted into one or more bovine V light chain loci. λ Gene segment coding sequence, one or more bovine J λ gene segment coding sequences and one or more rodent C λ gene, where V λ and J. λ Gene segment coding sequences and rodent (C λ ) region gene is integrated into the non-coding regulatory or scaffolding sequences of the rodent immunoglobulin kappa light chain locus.

[0166] In some embodiments, one or more bovine V λThe gene segment coding sequence may be derived from one or more rodent C λ One or more Js located upstream of the gene λ located upstream of the gene segment coding sequence, wherein V λ and J. λ Gene segment coding sequences and rodent C λ In some embodiments, the gene is inserted into a rodent immunoglobulin kappa light chain locus. λ The gene segment coding sequence may be derived from one or more rodent C λ One or more Js located upstream of the gene λ located upstream of the gene segment coding sequence, wherein V λ and J. λ The gene segment coding sequences as well as the rodent Cλ gene are integrated into the non-coding regulatory or scaffolding sequences of the rodent immunoglobulin κ light chain locus.

[0167] In one embodiment, rodent C λ The coding sequence is derived from rodent C λ1 , C λ2 or C λ3 The coding sequence is selected from:

[0168] In some embodiments, the engineered immunoglobulin locus is a fusion protein comprising one or more rodent V κ gene segment coding sequences and one or more rodent J κ Each gene segment coding sequence is deleted and one or more bovine V λ A gene segment coding sequence and one or more J λ The gene segment coding sequence is replaced by a rodent C at the locus. κ The coding sequence is rodent C λ1 , C λ2 or C λ3 Transgenic rodents or rodent cells are provided that contain a rodent immunoglobulin κ locus replaced with a coding sequence.

[0169] In some embodiments, the engineered immunoglobulin variable region locus comprises one or more bovine V λa gene segment coding sequence and one or more JC units, wherein each JC unit is a bovine J λ In some embodiments, the engineered immunoglobulin variable region locus comprises one or more bovine V gene segment coding sequences and a rodent Cλ gene. λ a gene segment coding sequence and one or more JC units, wherein each JC unit is a bovine J λ Gene segment coding sequences and rodent C λ a region coding sequence, wherein V λ The gene segment coding sequence and JC unit are inserted into a rodent immunoglobulin kappa light chain locus. In some embodiments, the engineered immunoglobulin variable region locus is inserted into one or more bovine V λ a gene segment coding sequence and one or more JC units, wherein each JC unit is a bovine J λ Gene segment coding sequences and rodent C λ a coding sequence, wherein V λ The gene segment coding sequences and JC units are integrated into the non-coding regulatory or scaffolding sequences of the rodent immunoglobulin kappa light chain locus.

[0170] In some embodiments, one or more bovine V λ The gene segment coding sequence is located upstream of and in the same transcriptional direction as one or more JC units, where each JC unit is a bovine JC unit. λ Gene segment coding sequences and rodent C λ In one embodiment, one or more bovine V λ The gene segment coding sequence is located upstream of and in the same transcriptional direction as one or more JC units, where each JC unit is a bovine JC unit. λ Gene segment coding sequences and rodent C λ In some embodiments, the engineered immunoglobulin variable region locus comprises one or more bovine V coding sequences located upstream of one or more JC units. λ gene segment coding sequences, wherein each JC unit is a bovine J λ gene segment coding sequence and a rodent Cλ coding sequence, wherein V λThe gene segment coding sequence and JC unit are inserted into a rodent immunoglobulin kappa light chain locus. In some embodiments, the engineered immunoglobulin variable region locus is inserted into one or more bovine V λ The gene segment coding sequence is upstream of and in the same transcriptional direction as one or more JC units, wherein each JC unit is a bovine J λ gene segment coding sequence and a rodent Cλ coding sequence, wherein V λ The gene segment coding sequence and JC unit are integrated into the non-coding regulatory or scaffolding sequences of the rodent immunoglobulin κ light chain locus. λ The coding sequence is derived from rodent C λ1 , C λ2 or C λ3 The coding sequence is selected from:

[0171] In some embodiments, the engineered immunoglobulin locus is a bovine V κ The engineered immunoglobulin locus comprises a coding sequence and a rodent non-coding regulatory or scaffold sequence from a rodent immunoglobulin light chain variable region gene locus. In some embodiments, the engineered immunoglobulin locus is a bovine V κ or J κ The engineered immunoglobulin locus comprises a gene segment coding sequence and a rodent non-coding regulatory sequence or scaffold sequence from a rodent immunoglobulin light chain variable region gene locus. In some embodiments, the rodent non-coding regulatory sequence or scaffold sequence is from a rodent immunoglobulin λ light chain variable region gene locus. In some embodiments, the rodent non-coding regulatory sequence or scaffold sequence is from a rodent immunoglobulin κ light chain variable region locus. In some embodiments, the engineered immunoglobulin locus is from a bovine V κ and J. κ The engineered immunoglobulin locus comprises a gene segment coding sequence and a rodent non-coding regulatory or scaffold sequence from a rodent immunoglobulin kappa light chain variable region gene locus. In some embodiments, the engineered immunoglobulin locus is a bovine V κ and J. κThe gene segment coding sequence and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin lambda light chain variable region gene locus. In some embodiments, the partially bovine immunoglobulin locus comprises one rodent immunoglobulin C κ In some embodiments, the partially bovine immunoglobulin locus comprises one or more rodent immunoglobulin C coding sequences. λ In some embodiments, the partially bovine immunoglobulin locus comprises one or more bovine V coding sequences. κ and J. κ Gene segment coding sequence and one rodent immunoglobulin C κ In some embodiments, the engineered immunoglobulin locus comprises a bovine V coding sequence integrated into a rodent non-coding regulatory sequence or scaffold sequence of a rodent kappa light chain variable region gene locus. κ and J. κ Gene segment coding sequence and one rodent immunoglobulin C κ In some embodiments, the engineered immunoglobulin locus comprises a bovine V coding sequence integrated into a rodent non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin λ light chain variable region gene locus. κ and J. κ Gene segment coding sequence and one rodent immunoglobulin C κ Contains coding sequences.

[0172] Without wishing to be bound by theory, it is believed that inactivating or rendering non-functional the endogenous rodent κ light chain locus may increase expression of λ light chain immunoglobulins, in part, from bovine immunoglobulin loci. This has been shown to be the case in otherwise conventional mice in which the κ light chain locus is inactivated in the germline (Zon, et al. (1995) Subtle differences in antibody responses and hypermutation of λ light chains in mice with a disrupted κ constant region. Eur. J. Immunol. 25:2154-2162). In some embodiments, inactivating or rendering non-functional the endogenous rodent κ light chain locus may increase the relative amount of λ light chain-containing immunoglobulins relative to the amount of κ light chain-containing immunoglobulins produced by transgenic rodents or rodent cells.

[0173] In some embodiments, a transgenic rodent or rodent cell is provided in which the endogenous rodent immunoglobulin κ light chain locus is deleted, inactivated, or non-functional. In some embodiments, the endogenous rodent immunoglobulin κ light chain locus is a transgenic rodent or rodent cell ... κ Deletion or mutation of gene segment coding sequence; all endogenous rodent J κ Deletion or mutation of gene segment coding sequence; endogenous rodent C κ Deletion or mutation of the coding sequence; endogenous intronic kappa enhancer (iE κ ) and a 3' enhancer sequence (3'E κ ) deletion, mutation, or disruption; or a combination thereof, which renders the gene inactivated or non-functional.

[0174] In some embodiments, a transgenic rodent or rodent cell is provided in which the endogenous rodent immunoglobulin λ light chain variable domain is deleted, inactivated, or non-functional. In some embodiments, the endogenous rodent immunoglobulin λ light chain variable domain is a λ light chain variable domain derived from the following all endogenous rodent V κGene segment deletion or mutation; all endogenous rodent J λ Gene segment deletion or mutation; all endogenous rodent C λ It may be rendered inactivated or non-functional by one or more of the following: deletion or mutation of the coding sequence; or a combination thereof.

[0175] In some embodiments, the partially bovine immunoglobulin locus comprises rodent regulatory or scaffold sequences, including but not limited to enhancers, promoters, splice sites, introns, recombination signal sequences, and combinations thereof. In some embodiments, the partially bovine immunoglobulin locus comprises rodent lambda regulatory or scaffold sequences. In some embodiments, the partially bovine immunoglobulin locus comprises rodent kappa regulatory or scaffold sequences.

[0176] In some embodiments, the partial bovine immunoglobulin locus comprises a promoter for driving gene expression. In some embodiments, the partial bovine immunoglobulin locus comprises a kappa V region promoter. In some embodiments, the partial bovine immunoglobulin locus comprises a lambda V region promoter. In some embodiments, the partial bovine immunoglobulin locus comprises a V λ From J λ and a λ V region promoter for driving expression of one or more λ LC gene coding sequences created after gene segment rearrangement. In some embodiments, the partially bovine immunoglobulin locus comprises a V κ From J κ and a λ V region promoter for driving expression of one or more κLC gene coding sequences created after gene segment rearrangement. In some embodiments, the partially bovine immunoglobulin locus comprises a V λ From J λ and a κ V region promoter for driving one or more λLC gene coding sequences created after gene segment rearrangement. In some embodiments, the partially bovine immunoglobulin locus comprises a V κ From J κ It includes a κ V region promoter for driving expression of one or more κ LC gene coding sequences created after gene segment rearrangement.

[0177] In some embodiments, the partially bovine immunoglobulin locus comprises one or more enhancers. In some embodiments, the partially bovine immunoglobulin locus comprises one or more enhancers. κ or 3'E κ In some embodiments, the partially bovine immunoglobulin locus comprises one or more V λ or J λ Gene segment coding sequence and moue κiE κ or 3'E κ In some embodiments, the partially bovine immunoglobulin locus comprises one or more V κ or J κ Gene segment coding sequence and κiE κ or 3'E κ Contains an enhancer.

[0178] Immunoglobulin heavy chain locus In some embodiments, the transgenic rodent or rodent cell contains a recombinantly produced, partially bovine immunoglobulin heavy chain variable region (V H In some embodiments, the partially bovine immunoglobulin variable region locus comprises one or more bovine V H , D or J H In some embodiments, the partially bovine immunoglobulin heavy chain variable region locus comprises one or more rodent constant domains (C H ) gene or coding sequence. In some embodiments, the endogenous rodent heavy chain immunoglobulin locus is inactivated. In some embodiments, the endogenous rodent heavy chain immunoglobulin locus is deleted and replaced with an engineered partial bovine heavy chain immunoglobulin locus.

[0179] In some embodiments, the synthetic heavy chain DNA segment comprises the ADAM6A or ADAM6B gene required for male fertility, a Pax-5-activated intergenic repeat (PAIR) element involved in Igh locus contraction, and a CTCF binding site from heavy chain intergenic regulatory region 1 involved in normal VDJ rearrangement regulation (Proudhon, et al., Adv. Immunol., 128:123-182 (2015)), or various combinations thereof. The location of these endogenous non-coding regulatory and scaffolding sequences in the mouse IGH locus is set forth in FIG. 1, which includes, from left to right, approximately 100 functional heavy chain variable region gene segments (101); PAIR, a Pax-5-activated intergenic repeat involved in IGH locus contraction for VDJ recombination (102); ADAM6A or ADAM6B, a disintegrin and metallopeptidase domain 6A gene required for male fertility (103); and the most distal D gene segment, IGHD-5. Pre-D region (104) upstream of a 21,609-bp fragment of D; V H Intergenic control region 1 (IGCR1), which contains the CTCF insulator locus to control gene segment usage (106); D, a diverse gene segment (10–15, depending on the mouse strain) (105); and four linked J H Gene segment (107); E, an intronic enhancer involved in VDJ recombination μ (108); S, the μ switch region for isotype switching μ (109);8 heavy chain constant region gene:C μ , C δ , C γ3 , C γ1 , C γ2b , C2 γa / c , C ε and C α (110) and the 3' regulatory region (3'RR) that controls isotype switching and somatic hypermutation (111). Figure 1A is a modified version of a diagram from Proudhon, et al., Adv. Immunol., 128:123-182 (2015).

[0180] In some embodiments, the engineered partially bovine region integrated into the mammalian host cell is a known bovine V H In some cases, however, such V H It may be desirable to use a subset of gene segments, and in certain cases, as few as one bovine V H The coding sequence can be introduced into a cell or an animal.

[0181] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a V nucleotide sequence from the bovine genome. H V containing most or all of the gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to 12 functional bovine V loci. H V containing gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from the bovine genome. H V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from Bos Taurus. H V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence H Includes sitting position.

[0182] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a V nucleotide sequence from the bovine genome. H V containing most or all of the gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and up to 12 bovine V loci. H V containing gene segment coding sequence HIn this embodiment, V H The gene segment pseudogenes are reverted to restore functionality, for example, by mutating in-frame stop codons to functional codons using methods well known in the art. In some embodiments, the engineered partially bovine immunoglobulin variable region loci are constructed from V sequences from the bovine genome. H V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence H Includes sitting position.

[0183] In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises a V gene segment coding sequence that includes most or all of the D gene segment coding sequence found in the bovine genome. H In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and up to 23 bovine D gene segment coding sequences. H In some embodiments, the engineered, partially bovine immunoglobulin variable region locus comprises a V locus that contains at least about 50%, 60%, 70%, 80%, 90%, and up to 100% D gene segment coding sequences found in the bovine genome. H In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus that contains at least about 50%, 60%, 70%, 80%, 90%, and up to 100% D gene segment coding sequences from bovine (Bos Taurus). H Includes sitting position.

[0184] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a J nucleotide sequence found in the bovine genome. H V containing most or all of the gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin locus variable region comprises at least one, two, or three, and up to four functional bovine J loci. HV containing gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a J locus found in the bovine genome. H V comprising at least about 50%, 75% and up to 100% of the gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a J locus from Bos Taurus. H V comprising at least about 50%, 75% and up to 100% of the gene segment coding sequence H Includes sitting position.

[0185] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is a V nucleotide sequence from the bovine genome. H , D and J H V containing most or all of the gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from the bovine genome. H , D and J H V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence H In some embodiments, the engineered partially bovine immunoglobulin variable region locus comprises a V locus from Bos Taurus. H , D and J H V that contains at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the gene segment coding sequence H Includes sitting position.

[0186] In some embodiments, a transgenic rodent or rodent cell is provided that comprises an engineered, partially bovine immunoglobulin heavy chain locus that includes bovine immunoglobulin heavy chain variable region gene coding sequences and non-coding regulatory or scaffolding sequences of a rodent immunoglobulin heavy chain locus. In some embodiments, the engineered bovine immunoglobulin heavy chain locus is a bovine V H , D or J HIn some embodiments, the engineered bovine immunoglobulin heavy chain locus comprises a bovine V gene segment coding sequence integrated into the non-coding regulatory or scaffold sequence of a rodent immunoglobulin heavy chain locus. H , D or J H Contains gene segment coding sequences.

[0187] In one embodiment, bovine V H , Ushi D and Ushi J H Non-bovine mammals and mammalian cells are provided that contain an engineered, partially bovine immunoglobulin locus that includes the coding sequence of a gene, and further contain non-coding regulatory and scaffolding sequences that include pre-D sequences based on the endogenous IGH locus of the non-bovine mammalian host. In some embodiments, the exogenously introduced, engineered, partially bovine region can include a fully recombined V(D)J exon.

[0188] In some embodiments, the transgenic non-bovine mammal comprises multiple bovine V H , Ushi D and Ushi J H A rodent, e.g., a mouse, that contains an exogenously introduced, engineered, partially bovine immunoglobulin locus, including intervening sequences that include the pre-D region, based on the codons of the gene and intervening (non-coding regulatory or scaffolding) sequences in the rodent. In one embodiment, the transgenic rodent contains a bovine V and VG locus, respectively, along with intervening (non-coding regulatory or scaffolding) sequences that correspond to the immunoglobulin intervening sequences present in the rodent IGL locus. κ or V λ Gene and J κ or J λ It further includes a partial bovine IGL locus that contains the coding sequence of the gene.

[0189] In an exemplary embodiment, as further detailed in the Examples section, the endogenous V H The entire immunoglobulin locus was deleted, followed by the J558 V site of the mouse genome. H Twenty bovine Vs containing interspersed non-coding sequences corresponding to the non-coding sequences of the locus HThe complete, exogenously introduced, engineered immunoglobulin locus is replaced with a partial bovine immunoglobulin locus containing gene segments, further comprising bovine D and J H It contains the gene segment as well as the mouse pre-D region. H , D and J H The codon sequences are incorporated into rodent intergenic and intron sequences.

[0190] Preparation of a partial bovine immunoglobulin locus. In one embodiment, V H , D and J H or V L and J. L Endogenous immunoglobulin locus variable regions of non-bovine mammals, such as rodents, e.g., rats or mice, containing gene segments are deleted using site-specific recombinases and replaced with engineered partial bovine immunoglobulin loci. In some embodiments, the partial bovine immunoglobulin loci are inserted into the host animal genome as a single nucleic acid or cassette. A cassette containing a partial bovine immunoglobulin locus is used to replace the endogenous immunoglobulin locus variable region, and the bovine coding sequence is inserted into the host genome in a single insertion step, thus providing a rapid and straightforward process for obtaining transgenic animals.

[0191] In some embodiments, the engineered partially bovine immunoglobulin locus variable region is derived from a mouse immunoglobulin locus variable region. H , D and J H or V L and J. L It is prepared by deleting the coding sequences and replacing the mouse coding sequences with bovine coding sequences, hi some embodiments, the non-coding flanking sequences of the mouse immunoglobulin loci, including regulatory sequences and other elements, remain intact.

[0192] In some embodiments, the nucleotide sequence for an engineered, partially bovine immunoglobulin locus is prepared in silico, and the locus is synthesized using known techniques for gene synthesis. In some embodiments, the coding sequence from the bovine immunoglobulin variable region locus and the sequence of the host animal immunoglobulin locus are identified using a search tool such as BLAST (Basic Local Alignment Search Tool). After obtaining the genomic sequence of the host immunoglobulin locus and the coding sequence of the bovine immunoglobulin variable region locus, the host coding sequence can be replaced in silico with the bovine coding sequence using known computational approaches to locate and delete the endogenous host animal immunoglobulin coding sequence and replace it with the coding sequence of the bovine coding sequence, leaving the endogenous regulatory and flanking sequences intact.

[0193] Homologous recombination In some embodiments, a combination of homologous recombination and site-specific recombination is used to generate the cells and animals described herein. In some embodiments, a homology targeting vector is first used to introduce a sequence-specific recombination site into the genome of a mammalian host cell at a desired location in an endogenous immunoglobulin locus. In some embodiments, the sequence-specific recombination site inserted into the genome of a mammalian host cell by homologous recombination in the absence of a recombinase protein does not affect the expression and amino acid codons of any genes in the mammalian host cell. This approach maintains proper transcription and translation of the immunoglobulin genes producing the desired antibody, and, optionally, any additional sequences, such as selectable marker genes, after insertion of the recombination site. However, in some cases, recombinase sites and other sequences can be inserted into the immunoglobulin locus sequence such that the amino acid sequence of the antibody molecule is altered by the insertion, but the antibody still retains sufficient functionality for the desired purpose. Examples of such codon-altering homologous recombination can include the introduction of polymorphisms into the endogenous locus and alteration of constant region exons so that a different isotype is expressed from the endogenous locus. In certain embodiments, the immunoglobulin locus comprises one or more such insertions.

[0194] In some embodiments, a homology targeting vector can be used to replace a specific sequence in an endogenous genome and insert a specific sequence-specific recombination site and one or more selectable marker genes into the host cell genome. As used herein, it will be understood by those skilled in the art that selectable marker genes can be used to eliminate individual cells that have not undergone homologous recombination and cells that have randomly integrated the targeting vector.

[0195] Exemplary methods of homologous recombination are described in US Patents 6,689,610; 6,204,061; 5,631,153; 5,627,059; 5,487,992; and 5,464,764, each of which is incorporated herein by reference in its entirety.

[0196] Site / Sequence-Specific Recombination Site / sequence-specific recombination differs from general homologous recombination in that a short, specific DNA sequence required for recognition by a recombinase is the only site at which recombination occurs. Depending on the orientation of these sites on a particular DNA strand or chromosome, specialized recombinases that recognize these specific sequences can catalyze i) DNA excision or ii) DNA inversion or rotation. Site-specific recombination can also occur between two DNA strands if these locations do not reside on the same chromosome. Numerous bacteriophage- and yeast-derived site-specific recombination systems, each containing a recombinase and a specific cognate site, have been shown to function in eukaryotic cells and are therefore applicable for use in connection with the methods described herein. These include bacteriophage P1 Cre / lox, the yeast FLP-FRT system, and the Dre system of the tyrosine family of site-specific recombinases. Such systems and methods of use are described, for example, in U.S. Patents 7,422,889; 7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and 4,959,317, each of which is incorporated herein by reference for its teachings of how to use such recombinases.

[0197] Other members of the tyrosine family of site-specific recombinases, such as bacteriophage lambda integrase, HK2022 integrase, and even members of the serine family of recombinases, such as bacteriophage phiC31, R4Tp901 integrase, are known to function in mammalian cells using their respective recombination sites and are applicable for use in the methods described herein.

[0198] Because site-specific recombination can occur between two different DNA strands, site-specific recombination events can be used as a mechanism to introduce exogenous loci into a host cell genome through a process called recombinase-mediated cassette exchange (RMCE). The RMCE process can be exploited by the combined use of wild-type and mutant sequence-specific recombination sites for the same recombinase protein along with negative selection. For example, a chromosomal locus to be targeted can be flanked by wild-type LoxP sites on one end and mutant LoxP sites on the other end. Similarly, an exogenous vector containing a sequence to be inserted into a host cell genome can similarly be flanked by wild-type LoxP sites on one end and mutant LoxP sites on the other end. When this exogenous vector is transfected into a host cell in the presence of Cre recombinase, Cre recombinase catalyzes RMCE between the two DNA strands rather than excision of the same DNA strand, because the wild-type and mutant LoxP sites on each DNA strand are incompatible for recombination with each other. Thus, a LoxP site in one DNA strand recombines with a LoxP site in the other DNA strand; similarly, a mutated LoxP site in one DNA strand recombines with a similarly mutated LoxP site in the other DNA strand.

[0199] In some embodiments, a combination of variants of sequence-specific recombination sites recognized by the same recombinase is used for RMCE. Examples of such sequence-specific recombination site variants include those that contain a combination of inverted repeats or those that contain recombination sites with mutant spacer sequences. For example, two classes of variant recombinase sites are available for designing stable Cre-loxP integrative recombination. Both utilize sequence mutations in the Cre recognition sequence within the 8bp spacer region or the 13bp inverted repeat. Spacer mutants such as lox511 (Hoess, et al., Nucleic Acids Res, 14:2287-2300 (1986)), lox5171 and lox2272 (Lee and Saito, Gene, 216:55-65 (1998)), m2, m3, m7, and m11 (Langer, et al., Nucleic Acids Res, 30:3067-3077 (2002)), while readily recombining with themselves, have significantly reduced rates of recombination with the wild-type position. This class of mutants has been utilized for DNA insertion by RMCE using non-interacting Cre-Lox and FLP recombination loci (Baer and Bode, Curr Opin Biotechnol, 12:473-480 (2001); Albert, et al., Plant J, 7:649-659 (1995); Seibler and Bode, Biochemistry, 36:1740-1747 (1997); Schlake and Bode, Biochemistry, 33:12746-12751 (1994)).

[0200] Inverted repeat mutants represent a second class of variant recombinase loci. For example, LoxP loci can contain modified bases in the left inverted repeat (LE mutant) or right inverted repeat (RE mutant). The LE mutant, lox71, has 5 bp at the 5' end of the left inverted repeat changed from the wild-type sequence to TACCG (Araki, et al., Nucleic Acids Res, 25:868-872 (1997)). Similarly, the RE mutant, lox66, has the 3'-most 5 bases changed to CGGTA. Inverted repeat mutants are used for integration of plasmid inserts into chromosomal DNA with LE mutants designated as "target" chromosomal loxP loci into which the "donor" RE mutant recombines. After recombination, the loxP loci are positioned in cis adjacent to the inserted segment. The mechanism of recombination is such that after recombination, one loxP locus is double mutant (containing both LE and RE inverted repeat mutations) and the other is wild-type (Lee and Sadowski, Prog Nucleic Acid Res Mol Biol, 80:1-42 (2005); Lee and Sadowski, J Mol Biol, 326:397-412 (2003)). The double mutant is sufficiently different from the wild-type locus that it is not recognized by Cre recombinase and the inserted segment is not excised.

[0201] In some embodiments, the sequence-specific recombination site can be introduced into an intron, as opposed to a coding nucleic acid region or regulatory sequence, to avoid inadvertently disrupting any regulatory sequences or coding regions necessary for proper antibody expression upon insertion of the sequence-specific recombination site into the genome of the animal cell.

[0202] Introduction of sequence-specific recombination sites can be achieved by conventional homologous recombination techniques. Such techniques are described in reference books such as Sambrook and Russell (2001) (Molecular cloning: a laboratory manual 3rd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press) and Nagy, A. (2003) (Manipulating the mouse embryo: a laboratory manual, 3rd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Renault and Duchateau, Eds. (2013) (Site-directed insertion of transgenes. Topics in Current Genetics 23. Springer). Tsubouchi, H. Ed. (2011) (DNA recombination, methods and protocols. Humana Press).

[0203] Specific recombination into genome can be promoted by using vector design for positive or negative selection, as known in the art.To facilitate the identification of cells undergoing replacement reaction, appropriate gene marker system can be used, and cells can be selected, for example, by using selective tissue culture medium.However, to ensure that the genome sequence is substantially free of exogenous nucleic acid sequences at or adjacent to the two ends of the replacement interval, the marker system / gene can be desirably removed after selecting cells containing replaced nucleic acid.

[0204] In some embodiments, cells in which replacement of all or part of an endogenous immunoglobulin locus has occurred are negatively selected by exposure to a toxin or drug. For example, cells that retain HSV-TK expression can be selected for by the use of a nucleoside analog such as ganciclovir. In other embodiments, cells containing deletions of an endogenous immunoglobulin locus can be positively selected for by the use of a marker gene, which can be removed, if desired, after or as a result of a recombination event. A possible positive selection system can be based on the use of two non-functional portions of a marker gene, such as HPRT, that are brought together by a recombination event. These two portions are operably linked upon a successful replacement reaction, where the functionally rearranged marker gene is further flanked on either side by sequence-specific recombination sites (different from the sequence-specific recombination sites used in the replacement reaction) such that the marker gene can be excised from the genome using an appropriate site-specific recombinase.

[0205] The recombinase can be provided as a purified protein or expressed from a vector construct transiently transfected into a host cell or stably integrated into the host cell genome. Alternatively, the cells can be first used to generate transgenic animals, which can then be bred with animals expressing the recombinase.

[0206] Because the methods described herein take advantage of two or more sets of sequence-specific recombination sites within the engineered genome, multiple rounds of RMCE can be utilized to introduce partially bovine immunoglobulin variable region genes into the genome of a non-bovine mammalian host cell.

[0207] Although not yet routine for the insertion of large DNA segments, CRISPR-Cas technology is another method for introducing chimeric bovine Ig loci.

[0208] Production of transgenic animals In one embodiment, a method is provided for producing a transgenic animal, eg, a rodent such as a mouse, that contains an introduced partial bovine immunoglobulin locus.

[0209] In some embodiments, the host cells utilized to replace endogenous immunoglobulin genes are embryonic stem (ES) cells, which can then be used to produce transgenic mammals. In some embodiments, the host cells are cells of an early embryo. In some embodiments, the host cells are pronuclear stage embryos or zygotes. Thus, in some embodiments, the methods described herein further comprise isolating embryonic stem cells or cells of an early embryo, such as a pronuclear stage embryo or zygote, that contain the introduced partial bovine immunoglobulin loci, and using the ES cells to produce transgenic animals that contain the replaced partial bovine immunoglobulin loci.

[0210] How to use In some embodiments, methods for producing antibodies comprising bovine variable regions are provided. In some embodiments, the methods comprise providing a transgenic rodent or rodent cell described herein and isolating an antibody comprising a bovine variable region expressed by the transgenic rodent. In some embodiments, methods for producing monoclonal antibodies comprising a bovine variable region are provided. In some embodiments, the methods comprise providing B cells from a transgenic rodent or cell described herein, immortalizing the B cells, and isolating an antibody comprising a bovine variable domain expressed from the immortalized B cells.

[0211] In some embodiments, the antibodies expressed by the transgenic rodent or rodent cells comprise a bovine HC variable domain. In some embodiments, the antibodies expressed by the transgenic rodent or rodent cells comprise a mouse HC constant domain. They may be of any isotype: IgM, IgD, IgG1, IgG2a / c, IgG2b, IgG3, IgE, or IgA.

[0212] In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a bovine HC variable domain and a mouse HC constant domain. In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a bovine LC variable domain and a mouse LC constant domain. In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a bovine HC variable domain and a bovine LC variable domain and a mouse HC constant domain and a mouse LC constant domain.

[0213] In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a bovine λ LC variable domain. In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a mouse λ constant domain. In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a bovine λ LC variable domain and a mouse λ constant domain. In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a bovine κ LC variable domain. In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a mouse κ constant domain. In some embodiments, the antibody expressed by the transgenic rodent or rodent cell comprises a bovine κ LC variable domain and a mouse κ constant domain.

[0214] In some embodiments, methods for producing antibodies or antigen-binding fragments comprising bovine variable regions are provided. In some embodiments, the methods comprise providing a transgenic rodent or cell described herein and isolating an antibody comprising a bovine variable region expressed by the transgenic rodent or rodent cell. In some embodiments, the variable regions of the antibody expressed by the transgenic rodent or rodent cell are sequenced, and antibodies comprising bovine variable regions obtained from antibodies expressed by the transgenic rodent or rodent cell can be recombinantly produced using known methods.

[0215] In some embodiments, methods are provided for producing immunoglobulins specific to an antigen of interest. In some embodiments, the methods include immunizing a transgenic rodent described herein with an antigen and isolating immunoglobulins specific to the antigen expressed by the transgenic rodent or rodent cells. In some embodiments, the variable domains of antibodies expressed by the rodent or rodent cells are sequenced, and antibodies comprising bovine variable regions that specifically bind to the antigen of interest are recombinantly produced using known methods. In some embodiments, the recombinantly produced antibody or antigen-binding fragment comprises bovine HC and LC, kappa or lambda, constant domains.

[0216] Incorporation by Citation All references cited herein, including patents, patent applications, articles, textbooks, and the like, and the references cited therein, are hereby incorporated by reference in their entirety for all purposes, unless already stated. [Example]

[0217] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments described below are all or the only experiments performed. Those skilled in the art will recognize that numerous variations and modifications can be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments, therefore, are to be considered in all respects as illustrative and not restrictive.

[0218] Efforts have been made to ensure accuracy with respect to periods and numbers used (e.g., vectors, amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0219] The examples describe targeting by both 5' and 3' vectors adjacent to the recombination and introduction site of synthetic DNA. Those skilled in the art will recognize after reading this specification that 5' vector targeting may occur first, followed by 3', or 3' vector targeting may occur first, followed by 5' vector. In some situations, targeting may be performed simultaneously using a dual detection mechanism.

[0220] Example 1: Introduction of an engineered, partially bovine immunoglobulin variable region gene locus into the immunoglobulin heavy chain variable region gene locus of a non-bovine mammalian host cell genome An exemplary method illustrating the introduction of an engineered, partially bovine immunoglobulin locus into a genomic locus of a non-mammalian ES cell is shown in further detail in Figures 2-6. In Figure 2, a homology targeting vector (201) is provided that contains a puromycin phosphotransferase-thymidine kinase fusion protein (puro-TK) (203) flanked by two different recombinase recognition loci (e.g., FRT (207) and loxP (205) for Flp and Cre, respectively) and two different mutant loci (e.g., modified mutant FRT (209) and mutant loxP (211)) that lack the ability to recombine with their respective wild-type counterparts / loci (i.e., wild-type FRT (207) and wild-type loxP (205)). The targeting vector further includes a diphtheria toxin receptor (DTR) cDNA (217) for use in negative selection of cells containing the introduced construct. The targeting vector also optionally includes a visible marker such as green fluorescent protein (GFP) (not shown). Regions 213 and 215 encode endogenous, non-bovine V H It is homologous to the 5' and 3' portions, respectively, of a contiguous region (229) in an endogenous non-bovine locus that is 5' to the genomic region containing gene segment (219). H gene segment (219), pre-D region (221), D gene segment (223), J HA homology targeting vector (201) carrying the immunoglobulin locus (231) containing gene segments (225) and immunoglobulin constant gene region genes (227) is introduced into ES cells (202). The site-specific recombination sequence and DTR cDNA from the homology targeting vector (201) are inserted into the endogenous mouse V H It integrates into the non-bovine genome at position 5' of the gene locus (204), resulting in the genome structure shown at 233. ES cells that do not have the exogenous vector (201) integrated into their genome are selected (killed) by including puromycin in the culture medium; only ES cells that have the exogenous vector (201) stably integrated into their genome and constitutively express the puro-TK gene are resistant to puromycin.

[0221] Figure 3 illustrates effectively the same approach as Figure 2, except that an additional set of sequence-specific recombination sites has been added, e.g., a Rox site (331) and a modified Rox site (335) for use with Dre recombinase. In Figure 3, a homology targeting vector (301) is provided that contains a puro-TK fusion protein (303) flanked by wild-type recombinase recognition sites for FRT (307), loxP (305), and Rox (331) and mutant sites for Flp (309), Cre (311), and Dre (335) recombinases that lack the ability to recombine with the wild-type sites 307, 305, and 331, respectively. The targeting vector also contains a diphtheria toxin receptor (DTR) cDNA (317). Regions 313 and 315 are located within the endogenous mouse V H The homology targeting vector is homologous to the 5' and 3' portions of the contiguous region (329) in the endogenous non-bovine locus (339) that is 5' to the genomic region containing the gene segment (319). H gene segment (319), pre-D region (321), D gene segment (323), J H(325) gene segments and the constant region genes (327) are introduced into the mouse immunoglobulin locus (339) (302). The site-specific recombination sequence in the homology targeting vector (301) and the DTR cDNA (317) are inserted into the endogenous mouse V H It is integrated into the mouse genome at position 5′ of the gene locus ( 304 ), resulting in the genome structure shown at 333.

[0222] As shown in Figure 4, a second homology targeting vector (401) is provided that contains an optional hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (435) that can be used for positive selection of HPRT-deficient ES cells; a neomycin resistance gene (437); and recombinase recognition sites FRT(407) and loxP(405) for Flp and Cre, respectively, that are capable of recombining with the FRT(407) and loxP(405) sites that were previously integrated into the mouse genome from the first homology targeting vector. The first homology targeting vector is designed to target the endogenous mouse V H Position 5' of locus (419) also contains a mutant FRT site (409), a mutant loxP site (411), a puro-TK fusion protein (403), and a DTR cDNA. Regions 429 and 439 are located in the endogenous J H The homology targeting vector is homologous to the 5' and 3' portions, respectively, of the contiguous region (441) at the endogenous non-bovine locus downstream of gene segment (425) and upstream of constant region gene (427). H gene segment (419), pre-D region (421), D gene segment (423), J H The modified mouse immunoglobulin locus (431) containing the gene segment (425) and the constant region gene (427) is introduced (402). The site-specific recombination sequences (407, 405), HPRT gene (435), and neomycin resistance gene (437) of the homology targeting vector are integrated (404) into the mouse genome upstream of the endogenous mouse constant region gene (427), resulting in the genome structure shown in 433.

[0223] Once the recombination locus is integrated into the mammalian host cell genome, the endogenous region of the immunoglobulin domain is then subjected to recombination by introduction of a recombinase corresponding to the sequence-specific recombination locus integrated into the genome, e.g., Flp or Cre. Figure 5 illustrates a modified Igh locus in the mammalian host cell genome, containing two integrated DNA fragments. One fragment contains a mutant FRT locus (509), a mutant LoxP locus (511), a puro-TK gene (503), a wild-type FRT locus (507), and a wild-type LoxP locus (505), and a DTR cDNA (517). H The other DNA fragment, containing the HPRT gene (535), the neomycin resistance gene (537), the wild-type FRT locus (507), and the wild-type LoxP locus (505), is integrated upstream of the gene locus (519). H The 525 gene integrates downstream of the gene locus but upstream of the constant region gene (527). In the presence of Flp or Cre (502), the entire intervening sequence between the DTR gene (517), the endogenous IGH variable region gene loci (519, 521, 525), and the wild-type FRT or wild-type LoxP site containing the HPRT (535) and neomycin resistance (537) genes is deleted, resulting in the genome structure shown in 539. The process relies on a second targeting event occurring on the same chromosome, but not its homolog (i.e., in cis, not trans). If targeting occurs in cis as intended, the DTR gene, which confers sensitivity to diphtheria toxin in rodents, is absent (deleted) from the host cell genome, and the cells are not susceptible to negative selection by diphtheria toxin introduced into the culture medium after Cre- or Flp-mediated recombination. Similarly, ES cells carrying random integration of the first or second targeting vector are sensitive to diphtheria toxin due to the presence of an intact DTR gene.

[0224] ES cells that are insensitive to diphtheria toxin are then screened for deletion of the endogenous variable region gene locus. Primary screening methods for deleted endogenous immunoglobulin loci can be performed by Southern blotting or polymerase chain reaction (PCR), followed by confirmation by a secondary screening technique such as Southern blotting.

[0225] Figure 6 shows the heavy chain variable region domains and V H and J H The endogenous IGH locus (V H , D and J H Figure 1 shows the integration of an engineered partially bovine sequence into a non-bovine genome that has previously been modified to delete part of the partially bovine V H gene locus (619), endogenous non-bovine pre-D gene region (621), partial bovine D locus (623), partial bovine J locus H A site-specific targeting vector (629) containing the gene locus (625) and flanking mutant FRT (609), mutant LoxP (611), wild-type FRT (607), and wild-type LoxP (605) loci is introduced into host cells (602). Specifically, a partial bovine V H Locus (619) contains 20 bovine V sequences with intervening sequences based on the endogenous non-bovine genome sequence. H the pre-D region (621) contains a 21.6 kb mouse sequence with considerable homology to the corresponding region of the endogenous bovine IGH locus; the D gene locus (623) contains the codons of 10 D gene segments integrated into intervening sequences surrounding the endogenous non-bovine D gene segments; and the J H Locus (625) contains four bovine J genes integrated into intervening sequences based on the endogenous non-bovine genome. H The IGH locus (601) in the host cell genome contains the codons for the V gene segment, including the intervening sequence, as shown in Figure 5. H , D and J HThe entire gene segment has been previously modified to be deleted. As a result of this modification, the endogenous non-bovine host cell IGH locus (601) remains with the puro-TK fusion gene (603), flanked upstream by a mutant FRT locus (609) and a mutant LoxP locus (611) and downstream by wild-type FRT (607) and wild-type LoxP (605). By introducing the appropriate recombinase (604), the partially bovine immunoglobulin locus is integrated into the genome upstream of the endogenous non-bovine constant region gene (627), resulting in the genomic structure shown at 631.

[0226] Cow V H , D and J H The sequences of the gene segment coding regions are in Table 1.

[0227] Primary screening methods for the introduction of partial bovine immunoglobulin loci can be performed by Southern blotting or PCR, followed by confirmation by secondary screening methods such as Southern blotting. H , D and J H It is designed to detect the presence of the gene locus as well as the entire intervening sequence.

[0228] Example 2: Introduction of an engineered, partially bovine immunoglobulin variable region gene locus containing additional non-coding regulatory or scaffolding sequences into the immunoglobulin heavy chain variable region gene locus of a non-bovine mammalian host cell genome In one embodiment, the partially bovine immunoglobulin locus comprises the elements described in Example 1, but with additional non-coding regulatory or scaffolding sequences, e.g., sequences strategically added to introduce additional regulatory sequences to ensure desired spacing within the introduced immunoglobulin locus, ensure proper juxtaposition of certain coding sequences with other sequences adjacent to the replaced immunoglobulin locus, etc. Figure 7 shows the introduction of a second exemplary engineered partially bovine sequence into a modified non-bovine genome, produced as described in Figures 2-5 and in Example 1 above.

[0229] Figure 7 shows the heavy chain variable region domains as well as the endogenous V H and J H The endogenous non-bovine IGH locus (V) encodes the entire intervening sequence between the gene loci.H , D and J H 7 shows the introduction of an engineered partially bovine sequence into a mouse genome previously modified to delete a portion of the V. A site-specific targeting vector (731) containing an engineered partially bovine immunoglobulin locus to be introduced into a non-bovine host genome is introduced (702) into genomic region (701). H gene locus (719), mouse pre-D region (721), partial bovine D locus (723), partial bovine J locus H A targeting vector (731) containing the gene locus (725), PAIR element (741), and flanking mutant FRT (709), mutant LoxP (711), wild-type FRT (707), and wild-type LoxP (705) loci is introduced into host cells (702). Specifically, an engineered partially bovine V H The locus (719) contains 20 bovine V sequences, along with intervening sequences based on the endogenous non-bovine genome sequence. H the pre-D region (721) contains 21.6 kb of non-bovine sequence located upstream of the endogenous non-bovine genome; the D region (723) contains the codons of the 10 bovine D gene segments integrated into intervening sequences surrounding the endogenous non-bovine D gene segments; and H Locus (725) contains four bovine J genes integrated into an intervening sequence based on endogenous non-bovine genomic sequences. H The IGH locus (701) of the host cell genome contains the codons for the V gene segment, including the intervening sequence, as described in connection with FIG. H , D and J H The entire gene segment has been previously modified to detect the entire gene segment. This modification leaves the endogenous non-bovine IGH locus (701) with the puro-TK fusion gene (703) flanked by a mutant FRT locus (709) and a mutant LoxP locus (711) upstream and a wild-type FRT locus (707) and a wild-type LoxP locus (705) downstream. By introducing the appropriate recombinase (704), the engineered partial bovine immunoglobulin locus is integrated into the genome upstream of the endogenous mouse constant region gene (727), resulting in the genomic structure shown at 729.

[0230] Primary screening methods for the introduction of engineered partially bovine immunoglobulin regions can be performed by Southern blotting or PCR, followed by confirmation by secondary screening methods such as Southern blotting. H , D and J H It is designed to detect the presence of the gene locus as well as the entire intervening sequence.

[0231] Example 3: Introduction of an engineered, partially bovine immunoglobulin locus into the immunoglobulin heavy chain gene locus of the mouse genome A method for replacing a portion of the mouse genome with an engineered, partially bovine immunoglobulin locus is shown in Figure 8. This method uses the introduction of a first site-specific recombinase recognition sequence into the mouse genome, followed by the introduction of a second site-specific recombinase recognition sequence into the mouse genome. The two loci are located at the endogenous mouse V H , D and J H The entire cluster of flanking gene segments is deleted using the appropriate site-specific recombinase as described herein.

[0232] V at wild-type mouse immunoglobulin locus (801) H (815), D(817) and J H (819) The targeting vectors (803, 805) used to introduce site-specific recombinase sequences on either side of the gene segment cluster and upstream of the constant region gene (821) contain additional site-specific recombination sequences that are still efficiently recognized by the recombinase but are modified so that they do not recombine with unmodified positions. This mutant modified position (e.g., lox5171) is then inserted into the endogenous V H , D and J H After deletion of gene segment (802), the non-native piece of DNA is placed in a targeting vector for use in a second site-specific recombination event to move the non-native piece of DNA into the modified IGH locus by RMCE. In this example, the non-native DNA is a synthetic nucleic acid containing both bovine and non-bovine sequences (809).

[0233] To accomplish the method outlined above, two gene targeting vectors are constructed. One of the vectors (803) contains the most distal V H It contains mouse genomic DNA taken from the 5' end of the Igh locus upstream of the gene segment. H It contains mouse genomic DNA taken from a locus within the downstream gene segment.

[0234] The key features of the 5' vector (803) are, from 5' to 3', the following: a gene encoding the diphtheria toxin A (DTA) subunit under the control of a modified herpes simplex virus type I thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (823); the most distal V in the Igh locus; H The vector contained 4.5 kb of mouse genomic DNA mapped upstream of the gene segment (825); an FRT recognition sequence for Flp recombinase (827); a piece of genomic DNA containing the mouse Polr2a gene promoter (829); a translation initiation sequence (a methionine codon integrated into a "Kozak" consensus sequence, 835); a mutated loxP recognition sequence for Cre recombinase (lox5171) (831); a transcription termination / polyadenylation sequence (pA. 833); a loxP recognition sequence for Cre recombinase (837); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of thymidine kinase (pu-TK) under the transcriptional control of the promoter from the mouse phosphoglycerate kinase 1 gene (839); and 3 kb of mouse genomic DNA located near the 5' end of the vector and arranged in its native relative orientation (841).

[0235] The key characteristics of the 3' vector (805) are, from 5' to 3': H and C Hthe HPRT gene (845) under the transcriptional control of the mouse Polr2a gene promoter; the neomycin resistance gene (847) under the control of the mouse phosphoglycerate kinase 1 gene promoter; the loxP recognition sequence for Cre recombinase (837); 2.1 kb of mouse genomic DNA (849) located immediately downstream of the 3.7 kb mouse genomic DNA fragment located proximal to the 5' end of the vector and positioned in its natural relative orientation; and the gene encoding the DTA subunit under the transcriptional control of a modified herpes simplex virus type 1 thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (823).

[0236] Mouse embryonic stem (ES) cells (derived from C57B1 / 6NTac mice) are transfected by electroporation with the 3' vector (805) according to widely used methods. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that only cuts the prokaryotic plasmid sequence or the associated polylinker. Transfected cells are plated, and after approximately 24 hours, positive selection is performed for cells with the 3' vector integrated into their DNA using the neomycin analog drug G418. Negative selection is also performed for cells in which the vector has integrated into their DNA, but not by homologous recombination. Non-homologous recombination results in the retention of the DTA gene (823), which will kill the cell when the gene is expressed, whereas homologous recombination deletes the DTA gene because it is outside the region of the vector that is homologous to the mouse IGH locus. Drug-resistant ES cell colonies are visible after approximately one week and then physically picked. These selected colonies are disaggregated, replated into microwell plates, and cultured for several days, after which each cell clone is split so that some cells are frozen as an archive and the rest can be used to isolate DNA for analytical purposes.

[0237] DNA from ES cell clones is screened by PCR using a widely used gene targeting assay design. For this assay, one PCR oligonucleotide primer sequence maps outside the region of identity shared between the 3' vector (805) and genomic DNA, while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., the HPRT (845) or neomycin resistance (847) gene. By standard design, these assays detect DNA fragments that would only be present in ES cell clones derived from transfected cells that have undergone sufficiently rational homologous recombination between the 3' targeting vector and the endogenous mouse IGH locus. Two separate transfections are performed with the 3' vector (805). PCR-positive clones from the two transfections are selected for expansion and then further analyzed using Southern blot assays.

[0238] Southern blot assays are performed according to widely used methods using three probes and genomic DNA digested with multiple restriction enzymes, chosen so that the probe-digest combination can identify the structure of the target locus in the clone as appropriately modified by homologous recombination. One probe maps to the DNA sequence adjacent to the 5' side of the region of identity shared between the 3' targeting vector and the genomic DNA; the second probe maps 3' to the region of identity, but outside; and the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., to the HPRT (845) or neomycin resistance (847) gene. Southern blots are performed with one of the external probes and the neomycin or HPRT probe to identify the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the portion of the IGH locus correctly, i.e., mutated by homologous recombination with the 3' Igh targeting vector. The external probe detects the mutant fragment and also the wild-type fragment from the non-mutant copy of the immunoglobulin Igh locus on the homologous chromosome.

[0239] The karyotypes of PCR- and Southern blot-positive ES cell clones are analyzed using an in situ fluorescent hybridization method designed to distinguish between the most commonly occurring chromosomal abnormalities that occur in mouse ES cells. Clones with such abnormalities are excluded from further use. ES cell clones that are determined to have the correct predicted genomic structure based on the Southern blot data—and that have no detectable chromosomal abnormalities based on karyotype analysis—are selected for further use.

[0240] Accepted clones are then modified with the 5' vector (803) using methods and screening assays similar in design to those used with the 3' vector (805), except that puromycin selection is used instead of G418 / neomycin for selection. PCR assays, probes, and digests are also tailored to suit the genomic region to be modified by the 5' vector (805).

[0241] Clones of ES cells that are mutated in the expected manner by both the 3' and 5' vectors, i.e., double-targeted cells carrying both engineered mutations, are isolated after vector targeting and analysis. Clones must have undergone gene targeting on the same chromosome, not on a homologous chromosome (i.e., the engineered mutations made by the targeting vectors must be in cis on the same DNA strand, not in trans on another homologous DNA strand). Clones in the cis configuration are separated from those in the trans configuration by analytical methods such as fluorescent in situ hybridization of metaphase spreads using a probe that hybridizes to the novel DNA present in the two gene targeting vectors (803 and 805) between the arms of genomic identity. The two types of clones can also be distinguished from each other by analyzing the drug resistance phenotype of surviving clones using a "sib selection" screening method, in which clones are transfected with a vector expressing Cre recombinase, which deletes the pu-TK (839), HPRT (845), and neomycin resistance (847) genes if the targeting vector is integrated in cis, and then the number of colonies surviving under ganciclovir selection against the thymidine kinase gene introduced by the 5' vector (803) is compared, and a portion of cells from the clones are tested for resistance to puromycin or G418 / neomycin. Cells carrying the cis-configured mutation are approximately 10 times more likely to survive than cells carrying the trans-configuration. 3 This is expected to produce many ganciclovir-resistant clones. The majority of the resulting cis-derived ganciclovir-resistant clones will also be sensitive to both puromycin and G418 / neomycin, compared to trans-derived ganciclovir-resistant clones, which should retain resistance to both drugs. Dual-targeted clones of cells carrying the engineered mutations in the cis configuration at the heavy chain locus will be selected for further use.

[0242] Doubly targeted clones of cells are transiently transfected with a vector expressing Cre recombinase, as in the analytical experiments summarized above, and the transfected cells are subsequently placed under ganciclovir selection. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the predicted deletion between the two engineered mutations made by the 5' (803) and 3' (805) targeting vectors. In these clones, Cre recombinase causes recombination (802) to occur between the loxP sites (837) introduced into the heavy chain locus by the two vectors to create the genomic DNA configuration shown as 807. Because the LoxP sites are positioned in the same relative orientation in the two vectors, recombination results in the excision of a loop of DNA encompassing the entire genomic interval between the two LoxP sites. The loop does not contain an origin of replication and therefore does not replicate during mitosis and is therefore lost from cells as they proliferate. The resulting clones carry a deletion of the DNA originally located between the two loxP sites. Clones with the predicted deletion are selected for further use.

[0243] ES cell clones carrying a sequence deletion in one of the two homologous copies of the immunoglobulin heavy chain locus were cloned using a bovine V gene flanked by mouse regulatory and flanking sequences. H , D and J H The resulting mouse model is retransfected with a Cre recombinase expression vector (804) along with a piece of DNA (809) containing a partial bovine immunoglobulin heavy chain locus, including the gene coding region sequence. Important features of this piece of synthetic DNA (809) include: a lox5171 locus (831); a neomycin resistance gene open reading frame (847) lacking an initiator methionine codon but which is in frame and contiguous with the uninterrupted open reading frame at the lox5171 locus; an FRT locus (827); and 20 bovine V sequences, each with a bovine coding sequence integrated into the mouse non-coding sequence. H An array of heavy chain variable region genes (851); optionally, a 10 kb pre-D region from the mouse heavy chain locus (not shown); bovine V H , D and JH Ten bovine D gene segments (853) and four bovine J segments in which coding sequences are integrated into mouse noncoding sequences H A 58 Kb piece of DNA containing the gene segment (855); a loxP locus (837) in the reverse relative orientation to the lox5171 locus (831).

[0244] Transfected clones are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, in which the engineered partial bovine donor immunoglobulin locus (809) is integrated in its entirety into the deleted endogenous immunoglobulin heavy chain locus between the lox5171 (831) and loxP (837) sites to create the DNA region shown at 811. Only cells that have properly undergone RMCE have the ability to express the neomycin resistance gene (847), because the promoter (829) and initiator methionine codon (835) required for its expression are absent from the vector (809) but are already pre-existing in the host cell IGH locus (807). Remaining elements from the 5' vector (803) are removed by Flp-mediated recombination (806) in vitro or in vivo, resulting in the final bovine-based locus shown at 813.

[0245] G418-resistant ES cell clones are analyzed by PCR and Southern blot to determine that they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones with the expected genomic structure are selected for further use.

[0246] To generate partially ES cell-derived chimeric mice using standard methods, ES cell clones carrying partially bovine immunoglobulin heavy chain DNA (813) at the mouse heavy chain locus are microinjected into mouse blastocysts from the DBA / 2 strain. Male chimeric mice with the highest level of ES cell-derived contribution to their coats are selected for mating with female mice. The selected female mice are of the C57B1 / 6NTac strain and also carry a transgene encoding Flp recombinase expressed in the germline. Progeny from these matings are analyzed for the presence of the partially bovine immunoglobulin heavy chain locus and the loss of the FRT-flanked neomycin resistance gene created by the RMCE process. Mice carrying the partially bovine locus are used to establish mouse colonies.

[0247] Example 4: Introduction of an engineered, partially bovine immunoglobulin locus into the immunoglobulin kappa chain gene locus of the mouse genome Another method for partially replacing a portion of the mouse genome with a bovine immunoglobulin locus is shown in Figure 9. This method involves first substituting the endogenous V κ (915) and J κ (919) Introduction of a first site-specific recombinase recognition sequence into the mouse genome, which may be introduced 5' or 3' of a cluster of gene segments, followed by a V κ and J. κ This involves the introduction into the mouse genome of a second site-specific recombinase recognition sequence that flanks the entire locus, including the gene segment constant region gene (921). Using the appropriate site-specific recombinase, as described herein, the flanking region is deleted and then partially replaced with a bovine immunoglobulin locus.

[0248] V κ (915) and J κ (919) The targeting vector used to introduce site-specific recombination sequences on either side of the gene segment also contains additional site-specific recombination sequences that are still efficiently recognized by the recombinase but are modified so that they do not recombine with unmodified positions. This position is located at the V κ and J. κAfter deletion of the gene segment cluster, non-native DNA fragments are modified by RMCE. κ The non-native DNA is placed in a targeting vector to migrate into the locus. In this example, the non-native DNA is a bovine V gene integrated into mouse regulatory and flanking sequences. κ and J. κ It is a synthetic nucleic acid that contains a gene segment coding sequence.

[0249] To accomplish the method outlined above, two gene targeting vectors are constructed. One vector (903) contains the most distal V κ It contains mouse genomic DNA taken from the 5' end of the locus, upstream of the gene segment. κ It contains mouse genomic DNA taken from a locus downstream (3') of gene segment (919) and upstream of the constant region gene (921).

[0250] Key features of the 5' vector (903) include: a gene encoding the diphtheria toxin A (DTA) subunit under the control of a modified herpes simplex virus type 1 thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (923); 6 kb of mouse genomic DNA (925) mapped upstream of the distal-most variable region gene in the kappa chain locus; an FRT recognition sequence for Flp recombinase (927); a piece of genomic DNA containing the mouse Polr2a gene promoter (929); a translation initiation sequence (935) incorporating a methionine codon in a "Kozak" consensus sequence; and C a mutated loxP recognition sequence for the Cre recombinase (lox5171) (931); a transcription termination / polyadenylation sequence (933); a loxP recognition sequence for the Cre recombinase (937); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of thymidine kinase (pu-TK) under the transcriptional control of a promoter from the mouse phosphoglycerate kinase 1 gene (939); and 2.5 kb of mouse genomic DNA (941) that was mapped closely to the 6 kb sequence at the 5' end of the vector and arranged in its native relative orientation.

[0251] Important characteristics of the 3' vector (905) are: κ (919) and C κ (921) 6 Kb of mouse genomic DNA (943) mapping between the gene loci; a gene encoding human hypoxanthine-guanine phosphoribosyltransferase (HPRT) under the transcriptional control of the mouse Polr2a gene promoter (945); a neomycin resistance gene (947) under the control of the mouse phosphoglycerate kinase 1 gene promoter; a loxP recognition sequence for Cre recombinase (937); 3.6 Kb of mouse genomic DNA (949) mapping immediately downstream of the 6 Kb DNA fragment contained at the 5' end of the vector and with two fragments oriented in the same transcriptional direction as in the mouse genome; and a gene encoding the diphtheria toxin A (DTA) subunit under the control of a modified herpes simplex virus type 1 thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (923).

[0252] Following a widely used method, mouse embryonic stem (ES) cells derived from C57B1 / 6NTac mice are electroporated with the 3' vector (905). Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that only cuts the prokaryotic plasmid sequence or the associated polylinker. Transfected cells are plated, and approximately 24 hours later, cells with the 3' vector integrated into their DNA are positively selected using the neomycin analog drug G418. Negative selection is also performed for cells in which the vector has integrated into their DNA but not via homologous recombination. Non-homologous recombination results in the retention of the DTA gene, which will kill the cell if expressed, whereas homologous recombination results in the deletion of the DTA gene because it is outside the region of vector homology at the mouse IGK locus. Drug-resistant ES cell colonies become visible approximately one week later and are physically picked. These selected colonies are disaggregated, replated into microwell plates, and cultured for several days. Each cell clone is then split so that some cells are frozen as an archive and the rest can be used to isolate DNA for analytical purposes.

[0253] DNA from ES cell clones is screened by PCR using a widely used gene targeting assay design. For this assay, one PCR oligonucleotide primer sequence maps outside the region of identity shared between the 3' vector (905) and genomic DNA (901), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., the HPRT (945) or neomycin resistance (947) gene. By standard design, these assays detect DNA fragments that would only be present in ES cell clones derived from transfected cells that have undergone sufficiently rational homologous recombination between the 3' vector (905) and the endogenous mouse IGK locus. Two separate transfections are performed with the 3' vector (905). PCR-positive clones from the two transfections are selected for expansion and then further analyzed using Southern blot assays.

[0254] Southern blot assays are performed according to widely used methods, including three probes and genomic DNA digested with multiple restriction enzymes selected so that the probes and digests allow for conclusions about the structure of the target locus in the clone and whether it has been properly modified by homologous recombination. One of the probes maps to the DNA sequence immediately 5' of the region of identity shared between the 3' κ targeting vector (905) and the genomic DNA; the second probe also maps 3' to the region of identity, but outside the region; and the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., to the HPRT (945) or neomycin resistance (947) gene. Southern blots, detected by one of the external probes and the neomycin or HPRT gene probe, identify the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the portion of the κ locus that has been correctly mutated, i.e., by homologous recombination with the 3' κ targeting vector (905). The external probe detects both the mutant fragment and also the wild-type fragment from the non-mutant copy of the immunoglobulin κ locus on the homologous chromosome.

[0255] The karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescent hybridization method designed to distinguish from the most commonly occurring chromosomal abnormalities that occur in mouse ES cells. Clones with such abnormalities are excluded from further use. Karyotypically normal clones determined to have the correct predicted genomic structure based on the Southern blot data are selected for further use.

[0256] Acceptable clones are then modified with the 5' vector (903) using methods and screening assays similar in design to those used for the 3' vector (905), except that puromycin selection is used instead of G418 / neomycin for selection and the protocol is adjusted to accommodate genomic region modification with the 5' vector (903). The goal of the 5' vector (903) transfection experiment is to isolate clones of ES cells that are modified in the predicted manner by both the 3' vector (905) and the 5' vector (903), i.e., double-targeted cells that carry both engineered mutations. In these clones, Cre recombinase induces recombination (902) between the loxP sites introduced into the kappa locus by the two vectors, resulting in the genomic DNA configuration shown in 907.

[0257] Furthermore, clones must have undergone gene targeting on the same chromosome, not on homologous chromosomes; i.e., the engineered mutations made by the targeting vectors must be in cis on the same DNA strand, not in trans on another homologous DNA strand. Analytical methods such as fluorescent in situ hybridization of metaphase spreads using probes that hybridize to novel DNA present in the two gene targeting vectors (903 and 905) between the arms of genomic identity allow clones in the cis configuration to be separated from those in the trans configuration. The two types of clones can also be distinguished from each other by analyzing the drug resistance phenotype of surviving clones using a "sib selection" screening method, in which clones are transfected with a vector expressing Cre recombinase, which deletes the pu-Tk (939), HPRT (945), and neomycin resistance (947) genes if the targeting vector is integrated in cis, and then the number of colonies surviving under ganciclovir selection against the thymidine kinase gene introduced by the 5' vector (903) is compared, and a portion of cells from the clones are tested for resistance to puromycin or G418 / neomycin. Cells carrying the cis-configured mutation are approximately 10 times more likely to survive than cells carrying the trans-configuration. 3This is expected to produce many ganciclovir-resistant clones. The majority of the resulting cis-derived ganciclovir-resistant clones will also be sensitive to both puromycin and G418 / neomycin, compared to trans-derived ganciclovir-resistant clones, which should retain resistance to both drugs. Dual-targeted clones of cells carrying engineered mutations in the cis configuration at the kappa chain locus will be selected for further use.

[0258] Doubly targeted clones of cells are transiently transfected with a vector (902) expressing Cre recombinase, as in the analytical experiments summarized above, and the transfected cells are subsequently placed under ganciclovir selection. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the predicted deletion (907) between the two engineered mutations made by the 5' vector (903) and the 3' vector (905). In these clones, Cre recombinase allows recombination to occur between the loxP sites (937) introduced into the kappa chain locus by the two vectors. Because the LoxP sites are positioned in the same relative orientation in the two vectors, recombination results in the excision of a loop of DNA encompassing the entire genomic interval between the two LoxP sites. The loop does not contain an origin of replication and therefore does not replicate during mitosis and is therefore lost from the cell as the clone expands. The resulting clone carries a deletion of the DNA originally located between the two LoxP sites. Clones with the predicted deletion are selected for further use.

[0259] ES cell clones carrying a sequence deletion in one of the two homologous copies of the immunoglobulin κ chain locus are designated V κ (951) and J κ(955) The Cre recombinase expression vector was retransfected (904) with a piece of DNA (909) containing a partial bovine immunoglobulin kappa chain locus, including the gene segment coding sequence. Important features of this piece of synthetic DNA (designated "KK") include: a lox5171 locus (931); a neomycin resistance gene open reading frame (947, lacking the initiator methionine codon but in frame and contiguous with the uninterrupted open reading frame at lox5171 locus (931)); an FRT locus (927); and eight bovine V sequences, each with a bovine coding sequence integrated into the mouse non-coding sequence. κ An array of gene segments (951); optionally, a 13.5 Kb piece of genomic DNA (not shown) immediately upstream of the cluster of Jκ region gene segments in the mouse κ chain locus; three bovine Jκ region gene segments integrated into mouse non-coding DNA. κ A 2 Kb piece of DNA containing the regional gene segment (955); a loxP locus (937) in the reverse relative orientation to the lox5171 locus (931).

[0260] The sequences of the bovine Vκ and Jκ gene coding regions are shown in Table 2.

[0261] In a second independent experiment, another piece of partially bovine DNA (909) was used in place of the KK DNA. Important features of this DNA (designated "LK") include: a lox5171 position (931); a neomycin resistance gene open reading frame (947) lacking an initiator methionine codon but which is in frame and contiguous with the uninterrupted open reading frame at lox5171 position (931); an FRT position (927); and 25 functional bovine V sequences, each with bovine coding sequences embedded in mouse non-coding control or scaffold sequences. λ an array of variable region gene segments (951); optionally, a 13.5 Kb piece of genomic DNA (not shown) immediately upstream of the cluster of Jκ region gene segments of the mouse κ chain locus; bovine Jκ region gene segments integrated into mouse non-coding DNA; λa 2 Kb piece of DNA containing the regional gene segment (955); a loxP locus (937) in the reverse relative orientation to the lox5171 locus (931).

[0262] Transfected clones from the KK and LK transfection experiments were placed under G418 selection, which enriched for clones of cells that had undergone RMCE, in which bovine donor DNA (909) had been integrated in its entirety into the deleted immunoglobulin kappa chain locus between the lox5171 (931) and loxP (937) sites located by the 5' (903) and 3' (905) vectors, respectively. Only cells that had undergone proper RMCE were capable of expressing the neomycin resistance gene (947), because the promoter (929) and initiator methionine codon (935) required for its expression were absent from the vector (909) but were already present in the host cell IGH locus (907). The DNA region created using the KK sequence is shown in 911. The remaining elements from the 5′ vector ( 903 ) are removed by Flp-mediated recombination in vitro or in vivo ( 906 ), resulting in the final bovine-based light chain locus shown at 913 .

[0263] G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine that they have undergone the expected RMCE process without unwanted rearrangements or deletions. Both KK and LK clones with the expected genomic structure are selected for further use.

[0264] To generate partially ES cell-derived chimeric mice by standard methods, KK and LK ES cell clones carrying partially bovine immunoglobulin DNA at the mouse κ chain locus (913) are microinjected into mouse blastocysts from the DBA / 2 strain. Male chimeric mice with the highest level of ES cell-derived contributions to their coats are selected for mating with female mice. Female mice selected for use in mating are of the C57B1 / 6NTac strain and also carry a transgene encoding Flp recombinase expressed in the germline. Progeny from these matings are analyzed for the presence of partially bovine immunoglobulin κ or λ light chain loci and for the loss of the FRT-flanked neomycin resistance gene created by the RMCE process. Mice carrying partially bovine loci are used to establish colonies of KK and LK mice.

[0265] Mice bearing a partial bovine heavy chain locus, produced as described in Example 3, can be bred with mice bearing a bovine-based κ chain locus. Their offspring are then bred together in a scheme that ultimately produces mice homozygous for both bovine-based loci, i.e., bovine-based for heavy chain and κ. Such mice produce a partial bovine heavy chain with bovine variable domains and mouse constant domains. They also produce a partial bovine κ protein with a bovine κ variable domain and a mouse κ constant domain from the κ locus. Monoclonal antibodies recovered from these mice have a bovine heavy chain variable domain paired with a bovine κ variable domain.

[0266] Variations in breeding schemes include producing mice that are homozygous for the bovine-based heavy chain locus but heterozygous at the κ locus, with a KK bovine-based locus on one chromosome and an LK bovine-based locus on the other chromosome. Such mice produce partially bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partially bovine κ proteins containing a bovine κ variable domain and a mouse κ constant domain from one of the κ loci. From the other κ locus, they produce partially bovine λ proteins with a bovine λ variable domain and a mouse κ constant domain. Monoclonal antibodies recovered from these mice have bovine variable domains paired in some cases with bovine κ variable domains and in other cases with bovine λ variable domains.

[0267] Example 5: Introduction of an engineered, partially bovine immunoglobulin locus into the immunoglobulin λ chain gene locus of the mouse genome Another method for replacing a portion of the mouse genome with an engineered, partially bovine immunoglobulin locus is shown in Figure 10. This method involves λ3 , C λ3 , J λ1 and C λ1 V in the immediate vicinity of the λ gene cluster (1023) λx / V λ2 Upstream of gene segment (1013) and V λ1 The wild-type mouse immunoglobulin λ locus (1001)-V was cloned by homologous recombination using a targeting vector (1003) that shares identity with both loci downstream of gene segment (1017). λx / V λ2 Gene segment (1013), J λ2 / C λ2 Gene cluster (1015) and V λ1 A vector containing a deletion of approximately 194 Kb of DNA from gene segment (1017) was constructed by deleting 194 Kb of DNA from a non-native piece of DNA modified by RMCE (1004). λ The non-naturally occurring DNA is replaced with an element designed to allow subsequent site-specific recombination to migrate into the locus. In this example, the non-naturally occurring DNA is a synthetic nucleic acid containing both bovine and mouse sequences.

[0268] Key features of the gene targeting vector (1003) for achieving the 194 Kb deletion are: a negative selection gene, such as the gene encoding the A subunit of diphtheria toxin (DTA, 1059) or the herpes simplex virus thymidine kinase gene (not shown); λx / V λ2 4 kb of genomic DNA from the 5' end of the variable region gene segment (1025); FRT locus (1027); a piece of genomic DNA containing the mouse Polr2a gene promoter (1029); a translation initiation sequence (a methionine codon incorporated into a "Kozak" consensus sequence) (1035); a mutant loxP recognition sequence for Cre recombinase (lox5171) (1031); a transcription termination / polyadenylation sequence (1033); an open reading frame encoding a protein responsible for puromycin resistance (1037), where this open reading frame is on the antisense strand relative to the Polr2a promoter and its flanking translation initiation sequence and is followed by its own transcription termination / polyadenylation sequence (1033); and a Cre recombinase a loxP recognition sequence for the Polr2a recombinase (1039); a translation initiation sequence (a methionine codon incorporated into the "Kozak" consensus sequence) on the same antisense strand as the puromycin resistance gene open reading frame (1035); a chicken beta-actin promoter and cytomegalovirus early enhancer element (1041) oriented to support transcription of the puromycin resistance open reading frame, all initiating translation at a start codon downstream of the loxP site on the antisense strand relative to the Polr2a promoter and its flanking translation initiation sequences, and proceeding backward from the loxP site to the puromycin open reading frame; a mutant recognition site for the Flp recombinase known as the "F3" site (1043); and a J λ3 , C λ3 , J λ1 and C λ1 A piece of genomic DNA upstream of the gene segment (1045).

[0269] Using a widely used method, mouse embryonic stem (ES) cells derived from C57B1 / 6NTac mice are transfected (1002) by electroporation with a targeting vector (1003). Homologous recombination replaces the native DNA in a 196 Kb region with sequences from the targeting vector (1003), resulting in the genomic DNA configuration shown in 1005.

[0270] Before electroporation, vector DNA is linearized with a rare-cutting restriction enzyme that only cuts the prokaryotic plasmid sequence or the associated polylinker. Transfected cells are plated and, approximately 24 hours later, placed under positive drug selection using puromycin. Negative selection is also performed for cells in which the vector has integrated into their DNA but not by homologous recombination. Non-homologous recombination results in the retention of the DTA gene, which will kill the cell if expressed, whereas homologous recombination results in the deletion of the DTA gene because it is outside the region of vector homology at the mouse IGL locus. Drug-resistant ES cell colonies become visible after approximately one week and are then physically picked. These selected colonies are disaggregated, replated into microwell plates, and cultured for several days. Each cell clone is then divided, with some cells frozen for archival purposes and the remainder used for DNA isolation for analytical purposes.

[0271] DNA from ES cell clones is screened by PCR using widely practiced gene targeting assay designs. For these assays, one PCR oligonucleotide primer sequence maps outside the region of identity shared between the targeting vector and genomic DNA, while the other maps within the novel DNA between the two arms of genomic identity of the vector, e.g., the puro gene (1037). By standard design, these assays detect pieces of DNA that would only be present in ES cell clones derived from transfected cells that have undergone sufficiently rational homologous recombination between the targeting vector (1003) and native DNA (1001).

[0272] Six PCR-positive clones from transfection (1002) are selected for expansion and further analysis using a Southern blot assay, which contains three probes and genomic DNA from the clones introduced with multiple restriction enzymes, chosen so that the combination of probes and digests allows identification of whether the ES cell DNA has been appropriately modified by homologous recombination.

[0273] The karyotypes of six PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescent hybridization method designed to distinguish the most common chromosomal abnormalities occurring in mouse ES cells. Clones showing evidence of abnormalities are excluded from further use. Karyotypically normal clones, determined to have the correct predicted genomic structure based on the Southern blot data, are selected for further use.

[0274] ES cell clones carrying a deletion of one of the two homologous copies of the immunoglobulin λ chain locus were designated V λ , J λ and C λ The Cre recombinase expression vector is then retransfected (1004) with a piece of DNA (1007) containing a partial bovine immunoglobulin λ chain locus, including the mouse λ region gene segments. Important features of this piece of DNA (1007) are: a lox5171 position (1031); a neomycin resistance gene open reading frame (1047) lacking an initiator methionine codon but in-frame at the lox5171 position and contiguous with an uninterrupted open reading frame; an FRT position (1027); an array of 25 functional bovine λ region gene segments (1051), each with a bovine λ coding sequence integrated into mouse λ non-coding sequences; and a bovine J region gene segment (1051) containing a bovine J region gene segment (1051). λ Bovine J gene segments and non-coding sequences from mouse λ constant domain gene segments are integrated λ A JC-unit array (1055) with gene segments and mouse λ constant domain gene segments (bovine J λ The gene segment is Jλ1 , J λ2 , J λ3 , J λ4 , J λ5 , J λ6 and J. λ7 The mouse λ constant domain gene segment encodes C λ1 or C λ2 or C λ3 a mutation recognition site for Flp recombinase known as the "F3" site (1043); an open reading frame conferring hygromycin resistance (1057) located on the antisense strand to the immunoglobulin gene segment coding information in the construct; and a loxP site in the reverse relative orientation to the lox5171 site (1039).

[0275] Cow V λ and J. λ The sequences of the gene coding regions are shown in Table 3.

[0276] Transfected clones are placed under G418 or hygromycin selection, which enriches for clones of cells subjected to the RMCE process in which the partially bovine donor DNA is integrated entirely into the deleted immunoglobulin lambda chain locus between the lox5171 and loxP sites placed there by the gene targeting vector. The remaining elements from the targeting vector (1003) are removed by in vitro or in vivo FLP-mediated recombination (1006), resulting in the final bovine locus shown in 1011.

[0277] G418 / hygromycin-resistant ES cell clones are analyzed by PCR and Southern blotting to determine whether they have undergone the expected recombinase-mediated cassette exchange process without unwanted rearrangements or deletions. Clones with the expected genomic structure are selected for further use.

[0278] To generate partially ES cell-derived chimeric mice using standard methods, ES cell clones carrying partially bovine immunoglobulin DNA (1011) at the mouse lambda chain locus are microinjected into mouse blastocysts from the DBA / 2 strain. Male chimeric mice with the highest level of ES cell-derived contributions to their coats are selected for mating with female mice of the C57B1 / 6NTac strain carrying a transgene encoding Flp recombinase expressed in the germline. Offspring from these matings are analyzed for the presence of the partially bovine immunoglobulin lambda chain locus and the loss of the FRT-flanked neomycin resistance gene and the F3-flanked hygromycin resistance gene created during the RMCE process. Mice carrying the partially bovine loci are used to establish mouse colonies.

[0279] In one embodiment, mice containing a bovine-based heavy chain and κ locus (as described in Examples 3 and 4) are bred with mice carrying a bovine-based λ locus. Mice produced by this type of breeding scheme are homozygous for the bovine-based heavy chain locus and can be homozygous for the KK bovine-based locus or the LK bovine-based locus. Alternatively, they can be heterozygous at the κ locus, carrying a KK locus on one chromosome and an LK locus on the other chromosome. Each of these mouse strains is homozygous for the bovine-based λ locus. Monoclonal antibodies recovered from these mice have a bovine κ variable domain paired in some cases with a bovine κ variable domain and in other cases with a bovine λ variable domain. The λ variable domain is derived from the bovine-based LK locus or the bovine-based λ locus.

[0280] Example 6: Introduction of an engineered, partially bovine immunoglobulin minilocus into the mouse genome In certain other embodiments, the partially bovine immunoglobulin locus comprises a bovine variable domain minilocus such as that depicted in Figure 11, wherein the bovine V H In place of a partial bovine immunoglobulin locus containing all or substantially all of the gene segment coding sequences, a mouse immunoglobulin locus is substituted for a chimeric bovine V gene that has been determined to be functional, i.e., not pseudogene-coding. HGene segments, e.g., 1–20 bovine V H It is replaced with a minilocus (1119) containing the gene segment.

[0281] A site-specific targeting vector (1131) containing the partial bovine immunoglobulin locus to be integrated is introduced (1102) into the genomic region (1101) containing a deletion of the endogenous immunoglobulin locus, including the puro-TK gene (1105) and the following flanking sequence-specific recombination sites: a mutant FRT site (1109), a mutant LoxP site (1111), a wild-type FRT site (1107), and a wild-type LoxP site (1105). The site-specific targeting vector contains: i) an array of any PAIR elements (1141); ii) e.g., 1-20 functional bovine V H V containing the coding region and an intervening sequence based on an endogenous sequence in the mouse genome H locus (1119); iii) a 21.6 kb pre-D region containing mouse sequences (1121); iv) 10 D and J loci H D locus (1123) and J, including an intervening sequence based on bovine coding sequence and endogenous sequence in the mouse genome H The partial bovine immunoglobulin locus contains a locus (1125). The partial bovine immunoglobulin locus is flanked by recombination sites - mutant FRT (1109), mutant LoxP (1111), wild-type FRT (1107), and wild-type LoxP (1105) - that allow for recombination with the modified endogenous locus. Upon introduction of an appropriate recombinase, e.g., Cre (1104), the partial bovine immunoglobulin locus is integrated into the genome upstream of the constant gene region (1127), as shown at 1129.

[0282] Primary screening for transfer of the partial bovine immunoglobulin variable region locus is performed by a primary PCR screen supported by a secondary Southern blotting assay, as described in Example 1. Deletion of the puro-TK gene (1105) as part of the recombination event allows for the identification of cells that have not undergone the recombination event using ganciclovir negative selection.

[0283] Example 7: Introduction of an engineered partial bovine immunoglobulin locus with bovine λ variable region coding sequences with mouse λ constant region sequences embedded in κ immunoglobulin non-coding sequences Bovine antibodies contain mostly λ light chains, while mouse antibodies contain mostly κ light chains. To increase the production of λLC-containing antibodies, endogenous mouse V κ and J. κ The Vκ region flanking and regulatory sequences of the LK mouse of Example 4 were integrated into the Vκ region. λ and J. λ The gene segment coding sequence is replaced with a bovine locus. In such mice, the endogenous regulatory sequences that drive high-level κ locus rearrangement and expression are expected to have an equivalent effect at the ectopic λ locus. However, in vitro studies have shown that the bovine V λ Domain is mouse C κ (See Example 9.) Therefore, the expected increase in λLC-containing antibodies in LK mice should not have occurred. κ and J. κ , Mouse V κ Replaced the flanking and regulatory sequences with mouse C κ Mouse C λ Incorporated V λ and J. λ The gene segment containing the coding sequence is replaced with a partial bovine locus.

[0284] Figure 13 shows one or more bovine V λ The gene segment coding sequence is selected from one or more rodent C λ one or more bovine J regions upstream of the coding sequence λ FIG. 1 is a schematic diagram illustrating the introduction of an engineered partial bovine light chain variable region locus inserted into a rodent immunoglobulin kappa light chain locus upstream of the gene segment coding sequence.

[0285] The method for partially replacing a portion of the mouse genome with a bovine immunoglobulin locus is shown in Figure 13. This method involves first substituting the endogenous V κ (1315) and J κ (1319) region of the cluster of gene segments 5' or 3' and C κ(1321) Introduction of a first site-specific recombinase recognition sequence into the mouse genome, which can be introduced into the exon, followed by a first sequence-specific recombination site in combination with the V κ and J. κ Clusters of gene segments and C κ This involves the introduction into the mouse genome of a second site-specific recombinase recognition sequence that flanks the entire locus, including the exons. Using the appropriate site-specific recombinase, as described herein, the flanking regions are deleted and then partially replaced with a bovine immunoglobulin locus.

[0286] V κ (1315) gene segment and C κ The targeting vector used to introduce the site-specific recombination sequences on either side of exon (1321) also contains additional site-specific recombination sequences that are still efficiently recognized by the recombinase but are modified so that they do not recombine with unmodified positions. κ and J. κ Gene segment clusters and C κ After exon deletion, the non-native piece of DNA is modified by RMCE. κ The non-native DNA is positioned on a targeting vector so that it can be used in a second site-specific recombination event to move into the locus. In this example, the non-native DNA is a bovine V integrated into the mouse IGK regulatory and flanking sequences. λ and J. λ Gene segment coding sequences and mouse C λ It is a synthetic nucleic acid containing exons.

[0287] To accomplish the method outlined above, two gene targeting vectors are constructed. One vector (1303) contains the most distal V κ It contains mouse genomic DNA taken from the 5' end of the locus, upstream of the gene segment. κ Taken from within the locus in regions spanning both upstream (5') and downstream (3') of exon (1321).

[0288] The key features of the 5' vector (1303) are: a gene encoding the diphtheria toxin A (DTA) subunit under the control of a modified herpes simplex virus type 1 thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (1323); 6 kb of mouse genomic DNA (1325) mapping upstream of the most distal variable region gene in the kappa chain locus; an FRT recognition sequence for Flp recombinase (1327); a piece of genomic DNA containing the mouse Polr2a gene promoter (1329); a translation initiation sequence (1335), incorporating a methionine codon in a "Kozak" consensus sequence; and a Cre gene. a mutated loxP recognition sequence for the recombinase (lox5171) (1331); a transcription termination / polyadenylation sequence (1333); a loxP recognition sequence for the Cre recombinase (1337); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of thymidine kinase (pu-TK) under the transcriptional control of a promoter from the mouse phosphoglycerate kinase 1 gene (1339); and 2.5 kb of mouse genomic DNA (1341), mapped near the 6 kb sequence at the 5' end of the vector and arranged in its native relative orientation.

[0289] Important characteristics of the 3' vector (1305) are: κ6 kb of mouse genomic DNA (1343) mapping within the locus in a region spanning the upstream (5') and downstream (3') exons (1321); a gene encoding human hypoxanthine-guanine phosphoribosyltransferase (HPRT) under the transcriptional control of the mouse Polr2a gene promoter (1345); a neomycin resistance gene (1347) under the control of the mouse phosphoglycerate kinase 1 gene promoter; a loxP recognition sequence for Cre recombinase (1337); 3.6 kb of mouse genomic DNA (1349) mapping immediately downstream of the 6 kb DNA fragment contained at the 5' end of the vector, with two fragments oriented in the same transcriptional direction as in the mouse genome; and a gene encoding the diphtheria toxin A (DTA) subunit under the control of a modified herpes simplex virus type 1 thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus (1323).

[0290] One strategy to delete the endogenous mouse IGK locus is to use the mouse C κ However, the 3' kappa enhancer, which is necessary for the modified locus to be retained, totals 9.6 kb, which is too short to be present in the upstream and downstream homology arms of the 3' vector. κ It is located 9.1 Kb downstream of the exon, therefore the upstream region of homology was expanded.

[0291] Using a widely used method, mouse embryonic stem (ES) cells derived from C57B1 / 6NTac mice are transfected with the 3' vector (1305) by electroporation with the 3' vector (1305). Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that only cuts the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and, after approximately 24 hours, placed under positive selection for cells with the 3' vector integrated into their DNA using the neomycin analog drug G418. There is also negative selection for cells in which the vector has integrated into their DNA, but not by homologous recombination. Non-homologous recombination retains the DTA gene, which kills cells when the gene is expressed, but the DTA gene is deleted by homologous recombination because it is outside the region of vector homology with the mouse IGK locus. Drug-resistant ES cell colonies are visible after approximately one week and then physically removed from the plate. These colonies are disaggregated, replated into microwell plates, and cultured for several days. Each clone of cells is then separated—some of the cells are frozen for archival purposes, while the rest are used to isolate DNA for analytical purposes.

[0292] DNA from ES cell clones is screened by PCR using a widely used gene targeting assay design. For this assay, one PCR oligonucleotide primer sequence maps outside the region of identity shared between the 3' vector (1305) and genomic DNA (1301), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., the HPRT (1345) or neomycin resistance (1347) gene. By standard design, these assays detect DNA fragments that would only be present in ES cell clones derived from transfected cells that have undergone sufficiently rational homologous recombination between the 3' vector (1305) and the endogenous mouse IGK locus. Two separate transfections are performed with the 3' vector (1305). PCR-positive clones from the two transfections are selected for expansion and then further analyzed using Southern blot assays.

[0293] This is performed according to commonly used methods, using three probes and genomic DNA digested with multiple restriction enzymes selected to allow for the determination of the structure of the clone's target locus and whether it was properly modified by homologous recombination. The first probe maps to the DNA sequence immediately 5' of the region of identity shared between the 3' κ targeting vector (1305) and the genomic DNA; the second probe also maps 3' outside the region of identity; and the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., to the HPRT (1345) or neomycin resistance (1347) gene. Southern blots, using one of the external probes and the neomycin or HPRT gene probe, identify the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the portion of the κ locus that was correctly mutated, i.e., mutated by homologous recombination with the 3' κ targeting vector (1305). The external probe detects both the mutant fragment and also the wild-type fragment from the non-mutant copy of the immunoglobulin κ locus on the homologous chromosome.

[0294] The karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescent hybridization method designed to distinguish from the most commonly occurring chromosomal abnormalities that occur in mouse ES cells. Clones with such abnormalities are excluded from further use. Karyotypically normal clones determined to have the correct predicted genomic structure based on the Southern blot data are selected for further use.

[0295] Acceptable clones are then modified with the 5' vector (1303) using methods and screening assays similar in design to those used for the 3' vector (1305), except that puromycin selection is used instead of G418 / neomycin for selection and the protocol is adjusted to accommodate genomic region modification with the 5' vector (1303). The goal of the 5' vector (1303) transfection experiment is to isolate clones of ES cells that are modified in the predicted manner by both the 3' vector (1305) and the 5' vector (1303), i.e., double-targeted cells that carry both engineered mutations. In these clones, Cre recombinase induces recombination (1302) between the loxP sites introduced into the kappa locus by the two vectors, resulting in the genomic DNA configuration shown in 1307.

[0296] Furthermore, clones must have undergone gene targeting on the same chromosome, not on a homologous chromosome; i.e., the engineered mutation created by the targeting vector must be in cis on the same DNA strand, not in trans on another homologous DNA strand. They can be distinguished from those in trans by analytical methods such as fluorescent in situ hybridization of metaphase spreads using a probe that hybridizes to the novel DNA present in the cis configuration. The two types of clones can also be distinguished from each other by transfecting them with a vector expressing Cre recombinase, which deletes the pu-Tk (1339), HPRT (1345), and neomycin resistance (1347) genes if the targeting vector is integrated in cis, comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5' vector (1303), and then analyzing the drug resistance phenotype of surviving clones using a "sib selection" screening method. This involves testing a portion of the clones for resistance to puromycin or G418 / neomycin. Cells with cis-configured mutations were approximately 10 times more likely to be mutated than cells with trans-configuration mutations. 3 This is expected to produce many ganciclovir-resistant clones. The majority of the resulting cis-derived ganciclovir-resistant clones will also be sensitive to both puromycin and G418 / neomycin, compared to trans-derived ganciclovir-resistant clones, which should retain resistance to both drugs. Dual-targeted clones of cells carrying engineered mutations in the cis configuration at the kappa chain locus will be selected for further use.

[0297] Doubly targeted clones of cells are transiently transfected with a vector (1302) expressing Cre recombinase, as in the analytical experiments summarized above, and the transfected cells are subsequently placed under ganciclovir selection. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the predicted deletion (1307) between the two engineered mutations made by the 5' vector (1303) and the 3' vector (1305). In these clones, Cre recombinase causes recombination between the loxP sites (1337) introduced into the kappa chain locus by the two vectors. Because the LoxP sites are positioned in the same relative orientation in the two vectors, recombination results in the excision of a loop of DNA encompassing the entire genomic interval between the two LoxP sites. The loop does not contain an origin of replication and therefore does not replicate during mitosis and is therefore lost from the cell as the clone expands. The resulting clone carries a deletion of the DNA originally between the two loxP sites and has the genomic structure shown in 1307. Clones carrying the predicted deletion are selected for further use.

[0298] ES cell clones carrying a sequence deletion in one of the two homologous copies of the immunoglobulin κ chain locus were designated V λ (1351) and J λ (1355) gene segment coding sequence and mouse C λ The DNA fragment (1309) containing a partial bovine immunoglobulin lambda chain locus, including exon (1357), was then retransfected with a Cre recombinase expression vector (1304). This synthetic DNA fragment contained the following key features: a lox5171 site (1331); a neomycin resistance gene open reading frame (1347, lacking the initiator methionine codon but in-frame at lox5171 site (1331) and contiguous with the uninterrupted open reading frame; an FRT site (1327); and 1 to 24 functional bovine V sequences, each with bovine coding sequences embedded within mouse non-coding control or scaffold sequences. λan array of variable region gene segments (1351); optionally, a 13.5 Kb piece of genomic DNA (not shown) immediately upstream of the cluster of Jκ region gene segments of the mouse κ chain locus; one to five functional bovine Jκ region gene segments integrated into the mouse non-coding DNA. λ A 2 Kb piece of DNA containing the region gene segment (1355) and mouse C λ exon (1357); loxP site (1337) in the reverse relative orientation to lox51 site (1331). The DNA fragment is deleted iE κ Also included (not shown).

[0299] Cow V λ and J. λ The sequences of the gene coding regions are shown in Table 3.

[0300] Transfected cells are placed under G418 selection, which partially enriches for clones of cells that have undergone RMCE, in which bovine donor DNA (1309) has been integrated in its entirety into the deleted immunoglobulin kappa chain locus between the lox5171 (1331) and loxP (1337) sites located by the 5' (1303) and 3' (1305) vectors, respectively. Only cells that have undergone RMCE properly have the ability to express the neomycin resistance gene (1347), because the promoter (1329) and initiator methionine codon (1335) required for its expression are absent from the vector (1309) but are already present in the host cell IGK locus (1307). The DNA region created by RMCE is shown in 1311. The remaining elements from the 5′ vector ( 1303 ) are removed by Flp-mediated recombination in vitro or in vivo ( 1306 ), resulting in the final bovine-based light chain locus shown at 1313 .

[0301] G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine that they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones with the expected genomic structure are selected for further use.

[0302] To generate partially ES cell-derived chimeric mice by standard methods, clones carrying partially bovine immunoglobulin DNA at the mouse kappa chain locus (1313) are microinjected into mouse blastocysts from the DBA / 2 strain. Male chimeric mice with the highest level of ES cell-derived contribution to their coats are selected for mating with female mice. Female mice selected for use in mating are of the C57B1 / 6NTac strain and also carry a transgene encoding Flp recombinase expressed in the germline. Progeny from these matings are analyzed for the presence of the partially bovine immunoglobulin lambda light chain locus and loss of the FRT-flanked neomycin resistance gene created by the RMCE process. Mice carrying the partially bovine locus are used to establish mouse colonies.

[0303] Mice bearing a partial bovine heavy chain locus, as produced as described in Example 3, can be bred with mice bearing a bovine λ-based κ-chain locus. Their offspring are then bred together in a scheme that ultimately produces mice homozygous for both bovine-based loci, i.e., bovine-based for both heavy and λ-based loci. Such mice produce a partial bovine heavy chain with bovine variable domains and mouse constant domains. They also produce a partial bovine λ protein with a bovine λ variable domain and a mouse λ constant domain from the κ-locus. Monoclonal antibodies recovered from these mice have a bovine heavy chain variable domain paired with a bovine λ variable domain.

[0304] Variations on the breeding scheme include producing mice that are homozygous for the bovine-based heavy chain locus but heterozygous at the κ locus, with the KK bovine-based locus described in Example 4 on one chromosome and the partially bovine λ-based κ locus described in this Example on the other chromosome. Such mice produce partially bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partially bovine κ proteins containing bovine κ variable domains and mouse κ constant domains from one of the κ loci. From the other κ locus, partially bovine λ proteins containing bovine λ variable domains and mouse λ constant domains are produced. Monoclonal antibodies recovered from these mice contain bovine κ variable domains in some cases and bovine variable domains paired with bovine λ variable domains in other cases.

[0305] Example 8. Introduction of an engineered partial bovine immunoglobulin locus with bovine λ variable region coding sequences with mouse λ constant region sequences embedded in mouse κ immunoglobulin non-coding sequences This example demonstrates the use of endogenous mouse V κ and J. κ , Mouse V κ Replaced the flanking and regulatory sequences with mouse C κ Mouse C λ Incorporated with Bovine V λ and J. λ Another strategy is described in Example 7, where a partial bovine locus containing gene segment coding sequence is replaced. However, in this example, the structure of the targeting vector containing the partial bovine locus is different. The bovine V gene locus coding sequence contains 1 to 24 functional V loci. λ Any array of gene segment coding sequences, followed by J λ -C λ an array of tandem cassettes, wherein J λ is of bovine origin, and C λ are of murine origin, e.g., C λ1 , C λ2 or C λ3 The number of cassettes is a unique functional bovine J λThe number of gene segments ranges from 1 to 5. The overall structure of the partially bovine λ locus in this example resembles the endogenous mouse λ locus, while the structure of the locus in Example 7 resembles the endogenous mouse κ locus that is partially replaced by the bovine λ locus in that example.

[0306] Figure 14 shows one or more bovine V λ Gene segment coding sequence J λ -C λ A schematic diagram illustrates an array of tandem cassettes inserted upstream of the rodent immunoglobulin kappa light chain locus, where J λ is of bovine origin, and C λ are of murine origin, e.g., C λ1 , C λ2 or C λ3 is.

[0307] The method for partially replacing a portion of the mouse genome with a bovine immunoglobulin locus is shown in Figure 14. This method replaces the endogenous V κ (1415) and J κ (1419) region of the cluster of gene segments 5' or 3' and C κ (1421) A first site-specific recombinase recognition sequence introduced into the exon was introduced into the mouse genome, followed by combining it with a first sequence-specific recombination site to form a V κ and J. κ Clusters of gene segments and C κ Introduction of a second site-specific recombinase recognition sequence into the mouse genome that flanks the entire locus, including the exons. Using the appropriate site-specific recombinase described herein, the flanking region is deleted and then partially replaced with a bovine immunoglobulin locus.

[0308] V κ (1415) gene segment and C κThe targeting vector used to introduce the site-specific recombination sequences on either side of exon (1421) also contains additional site-specific recombination sequences that are still efficiently recognized by the recombinase but are modified so that they do not recombine with unmodified positions. κ and J. κ Gene segment clusters and C κ After exon deletion, the non-native piece of DNA is modified by RMCE. κ The non-native DNA is positioned on a targeting vector so that it can be used in a second site-specific recombination event to move into the locus. In this example, the non-native DNA is a bovine V integrated into the mouse IGK regulatory and flanking sequences. λ Arrays of gene segment coding sequences and J λ is of bovine origin, and C λ are of murine origin, e.g., C λ1 , C λ2 or C λ3 J λ -C λ A synthetic nucleic acid comprising an array of tandem cassettes.

[0309] To accomplish the method outlined above, two gene targeting vectors are constructed. One vector (1403) contains the most distal V κ It contains mouse genomic DNA taken from the 5' end of the locus, upstream of the gene segment. κ Taken from within the locus in regions spanning both upstream (5') and downstream (3') of exon (1321).

[0310] Important characteristics of the 5' vector (1403) and 3' vector (1405) are as described in Example 7.

[0311] Mouse embryonic stem (ES) cells derived from C57B1 / 6NTac mice are transfected with the 3' vector (1405) by electroporation using a widely used method, as described in Example 7. DNA from ES cell clones is screened by PCR using a widely used gene targeting assay, as described in Example 7. Southern blot assays are performed using a widely used method, as described in Example 7.

[0312] The karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescent hybridization method designed to distinguish from the most commonly occurring chromosomal abnormalities that occur in mouse ES cells. Clones with such abnormalities are excluded from further use. Karyotypically normal clones determined to have the correct predicted genomic structure based on the Southern blot data are selected for further use.

[0313] Accepted clones are modified with the 5' vector (1403) using the methods and screening assays described in Example 7. The resulting correctly targeted ES clones are those in which the 5' vector (1403) is modified with the endogenous V κ The gene segment was inserted upstream, and the 3' vector (1405) was inserted into the endogenous C κ The clones have the genomic DNA configuration of the endogenous κ locus inserted downstream of 1407. In these clones, Cre recombinase induces recombination (1402) between the loxP sites introduced into the κ locus by the two vectors, resulting in the genomic DNA configuration shown in 1407.

[0314] Accepted clones are subjected to gene targeting on the same chromosome as opposed to the homologous chromosome, so that the engineered mutations made by the targeting vector are in cis on the same DNA strand, rather than in trans on a separate homologous DNA strand. Clones in the cis configuration are distinguished from those in the trans configuration by the analysis described in Example 7.

[0315] Doubly targeted clones of cells are transiently transfected with a vector expressing Cre recombinase (1402), as described in Example 7. Subsequently, the transfected cells are placed under ganciclovir selection and analyzed. In the selected clones, Cre recombinase allows recombination to occur between the loxP sites (1437) introduced into the kappa chain locus by the two vectors. Because the LoxP sites are positioned in the same relative orientation in the two vectors, recombination results in the excision of a loop of DNA encompassing the entire genomic interval between the two LoxP sites. The loop does not contain an origin of replication and therefore does not replicate during mitosis and is therefore lost from the cell as the clone expands. The resulting clone carries a deletion of the DNA originally located between the two LoxP sites and has the genomic structure shown in 1407. Clones with the predicted deletion are selected for further use, incorporating mouse IGK flanking and regulatory DNA sequences (1457).

[0316] ES cell clones carrying a sequence deletion in one of the two homologous copies of the immunoglobulin κ chain locus are designated V λ (1451) segment coding sequence and bovine J λ Gene segment coding sequences and mouse C λ The Cre recombinase expression vector was then retransfected (1404) with a piece of DNA (1409) containing a partial bovine immunoglobulin lambda chain locus containing a tandem array of exon-containing cassettes. Key features of this synthetic DNA piece include: a lox5171 site (1431); a neomycin resistance gene open reading frame (1447, lacking the initiator methionine codon but in-frame at the lox5171 site (1431) and contiguous with the uninterrupted open reading frame); an FRT site (1427); and 1 to 24 functional bovine V sequences, each containing bovine coding sequences embedded within mouse non-coding control or scaffold sequences. λan array of variable region gene segments (1451); optionally, a 13.5 Kb piece of genomic DNA (not shown) immediately upstream of the cluster of Jκ region gene segments of the mouse κ chain locus; a bovine Jκ region gene segment integrated with mouse IGK flanking and regulatory DNA sequences; λ Gene segment coding sequences and mouse C λ DNA containing a tandem array of exon-containing cassettes (1457); loxP locus (1437) in the reverse relative orientation to the lox5171 locus (1431).

[0317] Cow V λ and J. λ The sequences of the gene coding regions are shown in Table 3.

[0318] Transfected cells are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, containing the partial bovine donor DNA (1409) integrated entirely into the deleted immunoglobulin kappa chain locus between the 5' (1403) and 3' (1405) immunoglobulin kappa chain loci placed there by the vector, respectively. Only cells that have properly undergone RMCE are capable of expressing the neomycin resistance gene (1447), because the promoter (1429) and initiator methionine codon (1435) required for its expression are absent from the vector (1409) and are already pre-existing in the host cell IGK locus (1407). The DNA region created by RMCE is shown in 1411. The remaining elements from the 5′ vector ( 1403 ) are removed by Flp-mediated recombination in vitro or in vivo ( 1406 ), resulting in the final bovine-based light chain locus shown at 1413 .

[0319] G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine that they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones with the expected genomic structure are selected for further use.

[0320] To generate partially ES cell-derived chimeric mice by standard methods, clones carrying partially bovine immunoglobulin DNA at the mouse kappa chain locus (1413) are microinjected into mouse blastocysts from the DBA / 2 strain. Male chimeric mice with the highest level of ES cell-derived contribution to their coats are selected for mating with female mice. Female mice selected for use in mating are of the C57B1 / 6NTac strain and also carry a transgene encoding Flp recombinase expressed in the germline. Progeny from these matings are analyzed for the presence of the partially bovine immunoglobulin lambda light chain locus and loss of the FRT-flanked neomycin resistance gene created by the RMCE process. Mice carrying the partially bovine locus are used to establish mouse colonies.

[0321] Mice bearing a partial bovine heavy chain locus, produced as described in Example 3, can be bred with mice bearing a bovine λ-based κ chain locus. Their offspring are then bred together in a scheme that ultimately produces mice homozygous for both bovine-based loci, i.e., bovine-based for heavy chain and λ-based κ. Such mice produce a partial bovine heavy chain with bovine variable domains and mouse constant domains. They also produce a partial bovine λ protein with a bovine λ variable domain and a mouse λ constant domain from the κ locus. Monoclonal antibodies recovered from these mice have a bovine heavy chain variable domain paired with a bovine λ variable domain.

[0322] Variations on the breeding scheme include producing mice that are homozygous for the bovine-based heavy chain locus but heterozygous at the κ locus, with the KK bovine-based locus described in Example 4 on one chromosome and the partially bovine λ-based κ locus described in this Example on the other chromosome. Such mice produce partially bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partially bovine κ proteins containing bovine κ variable domains and mouse κ constant domains from one of the κ loci. From the other κ locus, they produce partially bovine λ proteins with bovine λ variable domains and mouse λ constant domains. Monoclonal antibodies recovered from these mice have bovine variable domains paired in some cases with bovine κ variable domains and in other cases with bovine λ variable domains.

[0323] The above method of introducing an engineered partially bovine immunoglobulin locus having bovine λ variable region coding sequences and mouse λ constant region sequences integrated into mouse κ immunoglobulin non-coding sequences is also applicable to mouse C κ Another method involves the deletion of an exon. κ This includes exon inactivation. Introns must be removed from primary mRNA transcripts through a process known as RNA splicing, in which the spliceosome, a large molecular machinery located in the nucleus, recognizes sequences at the 5' (splice donor) and 3' (splice acceptor) ends of the intron as well as other features of the intron, including the polypyrimidine tract located immediately upstream of the splice acceptor. The splice donor sequence in DNA is NGT, where "N" is any deoxynucleotide, and the splice acceptor is AGN (Cech TR, Steitz JA, and Atkins JF Eds. (2019) (RNA Worlds: New Tools for Deep Exploration, CSHL Press) ISBN 978-1-621822-24-0).

[0324] Mouse C κThe exon is inactivated by mutations in the splice acceptor sequence and the polypyrimidine tract. κ The wild-type sequence upstream of the exon is CTTCCTTCCTC AG (SEQ ID NO: 294) (splice acceptor site underlined). AAA TTAATTAA CC (SEQ ID NO: 295), resulting in a non-functional splice acceptor site and therefore a non-functional C κ This results in an exon. The mutant sequence also introduces a PacI restriction enzyme site (underlined). As an 8-base pair recognition sequence, this restriction site is predicted to be rare in the mouse genome (approximately every 65,000 bp), simplifying confirmation of whether the mutant sequence has been inserted into the IGK locus by Southern blot analysis of ES cell DNA digested with PacI and other frequent-cutting restriction enzymes. The wild-type sequence 3' is replaced with the mutant sequence by homologous recombination, a technique well known in the art for insertion of the RMCE vector. κ Important features of the homologous recombination vector (MSA, 1457) that mutates the exon splice acceptor sequence and polypyrimidine tract are: κ C instead of the wild-type CTTCCTTCCTCAG (SEQ ID NO: 294) sequence in its natural location immediately upstream of the exon κ6 Kb of mouse genomic DNA (1443) mapping to a region within the kappa locus spanning both upstream (5') and downstream (3') of exon (1421) and containing the mutant AAATTAATTAACC (SEQ ID NO: 295) (1459) sequence; a neomycin resistance gene (1447) under the control of the mouse phosphoglycerate kinase 1 gene promoter and flanked by mutant FRT sites (1461); 3.6 Kb of mouse genomic DNA (1449) mapping immediately downstream of the 6 Kb DNA fragment contained at the 5' end of the vector and carrying two fragments oriented in the same transcriptional direction as in the mouse genome; and a gene encoding the diphtheria toxin A (DTA) subunit (1423) under the control of a modified herpes simplex virus type 1 thymidine kinase gene promoter linked to two mutant transcriptional enhancers from polyomavirus. A mutant FRT locus (1461), such as FRT F3 or FRT F5 (Schlake and Bode (1994) Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33:12746-12751 PMID: 7947678 DOI: 10.1021 / bi00209a003), is used here because once the splicing mutation is introduced by transient transfection of an FLP recombinase expression vector (1406) to delete the Neo gene, the ES cells can be subjected to further genetic manipulation. This process requires the wild-type FRT locus (1447 at 1403) to delete the other Neo selection gene. If the FRT position (1461) remaining at the IGK locus (1469) after the introduction of the splicing mutation is wild-type, then this second attempt at FRT-mediated deletion of the Neo gene (1406 at 1413) would result in an entirely novel introduced, partially bovine locus and an inactivated mouse C. κ This can inadvertently result in the deletion of an exon.

[0325] Using a widely used method, mouse embryonic stem (ES) cells derived from C57B1 / 6NTac mice are transfected with the MSA vector (1457) by electroporation with the MSA vector (1457). Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that only cuts the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated, and after approximately 24 hours, they are placed under positive selection for cells with the MSA vector integrated into their DNA using the neomycin analog drug G418. There is also negative selection for cells in which the vector has integrated into their DNA, but not by homologous recombination. Non-homologous recombination results in the retention of the DTA gene, which will kill the cell if the gene is expressed, whereas homologous recombination results in the deletion of the DTA gene because it is outside the region of vector homology in the mouse IGK locus. Drug-resistant ES cell colonies become visible after approximately one week and are then physically picked. These selected colonies are disaggregated, replated into microwell plates, and cultured for several days, after which each clone of cells is split so that a portion of the cells are frozen for archival purposes and the remainder are used to isolate DNA for analytical purposes.

[0326] The IGK locus in ES cells that has been correctly targeted by homologous recombination has the configuration shown in 1463.

[0327] DNA from ES cell clones is screened by PCR using a widely used gene targeting assay design. For this assay, one PCR oligonucleotide primer sequence maps outside the region of identity shared between the MSA vector (1457) and genomic DNA (1401), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., the neomycin resistance (1447) gene. By standard design, these assays detect DNA fragments that would only be present in ES cell clones derived from transfected cells that have undergone sufficiently rational homologous recombination between the MSA vector (1457) and the endogenous mouse IGK locus. Two separate transfections are performed with the MSA vector (1457). PCR-positive clones from the two transfections are selected for expansion and then further analyzed using Southern blot assays.

[0328] Southern blot assays are performed using a widely used method, using three probes and genomic DNA digested with multiple restriction enzymes, chosen so that the combination of probes and digests allows for the determination of the structure of the target locus in the clone and whether it has been appropriately modified by homologous recombination. In this particular example, DNA is double-digested with PacI and another restriction enzyme, such as EcoRI or HindIII, because only cells into which the MSA vector has integrated contain a PacI locus. The first probe maps to the DNA sequence immediately 5' of the region of identity shared between the MSA vector (1457) and the genomic DNA; the second probe also maps outside the region of identity but 3'; and the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., the neomycin resistance (1447) gene. Southern blots are performed using one of the external probes and a neomycin resistance gene probe to precisely identify the presence of the predicted restriction enzyme-generated fragment of the corresponding DNA mutated by homologous recombination of the MSA κ targeting vector (1457) portion of the κ locus. The external probe detects the mutant fragment and also the wild-type fragment from the non-mutant copy of the immunoglobulin κ locus on the homologous chromosome. Southern blot assays are performed according to widely used methods described in Example 7.

[0329] The karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescent hybridization method designed to distinguish from the most commonly occurring chromosomal abnormalities that occur in mouse ES cells. Clones with such abnormalities are excluded from further use. Karyotypically normal clones determined to have the correct predicted genomic structure based on the Southern blot data are selected for further use.

[0330] The ability of ES cell DNA to be digested with PacI in the mutant IGK allele confirms the presence of the TTAATTAA sequence, but not the full predicted splicing mutation, C κDNA sequencing is performed focusing on the region upstream of the exon. The region is amplified by genomic PCR using primers flanking the mutation [1465 and 1467 (Table 5: SEQ ID NO: 275 and SEQ ID NO: 276)]. Another primer pair is shown in SEQ ID NO: 277 and SEQ ID NO: 278. These primers are designed using NCBI Primer-Blast and verified in silico to be free of any predicted off-target binding sites in the mouse genome.

[0331] Sequence-verified ES cell clones are transiently transfected with an FLP recombinase expression vector (1406) to delete the neomycin resistance gene (1427). Cells are then subcloned and the deletion is confirmed by PCR. The IGK locus in ES cells has the genomic arrangement shown in 1469.

[0332] ES cells are electroporated with the 5' and 3' RMCE vectors as described above, the only difference being that the 3' vector (1405) contains the mutant C κ The insertion of the exon upstream replaces the upstream and downstream homology arms of the 3' vector (1405) with sequences 943 and 949 of the 3' vector (905), respectively, as shown in Figure 9. Consequently, the PCR primers and Southern blot probes used to determine the correct integration of the 3' vector (1405) are derived from sequences 943 and 949 instead of 1443 and 1449. κ The enhancer is not included in the targeting vector (1409) as this sequence is not deleted.

[0333] Example 9: Bovine V λ The domain is mouse C κ Domain and does not function well, Bovine V κ The domain is mouse C λ It doesn't work well with the domain. For the proposed LK mouse (Example 4), a bovine V flanked by mouse non-coding and regulatory sequences was used. λ and J. λThe gene segment coding sequence is κ and J. κ The gene segment is integrated into the deleted mouse IGK locus. λ J λ After gene rearrangement, the resulting Ig gene encodes an LC with a bovine λ variable domain and a mouse κ constant domain. To test whether such hybrids can be properly expressed to form intact Ig molecules, a series of transient transfection assays were performed with various V combinations, V κ and V λ and C light chain exons, κ and C λ Both were performed with Ig HC to examine cell surface and intracellular expression and secretion of the encoded Ig.

[0334] For these experiments, bovine sequences encoding two bovine IGH variable region domains were tested, both of which were cloned from heterohybridomas and hypermutated in somatic cells. One was a mouse IgM b One contained an ultralong HCDR3 (clone BLV1H12, Accession # AF015506.1) linked to the allotype HC, while the other contained a normal-length HCDR3 (clone B4, Accession #U11628.1). H The coding DNA contained the endogenous bovine L1-intron-L2. Each was individually cloned into the pCMV vector. Bovine Vλ (AF023843.1) or Vκ (BC122795) V L Exon to mouse C κ , C λ1 , C λ2 or C λ3 The L1-intron-L2 sequences in each VL are all of bovine origin. These sequences contain hypermutated somatic residues that revert to germline sequences.

[0335] To test cell surface IgM expression, 293T / 17 cells were cotransfected with one of the HC and LC constructs and a CD79a / b expression vector, along with a human CD4 (hCD4) expression vector as a transfection control. The CD79a / b heterodimer was required for cell surface expression of IgM. Approximately 24 hours later, transfected cells were subjected to cell surface or intracellular staining by flow cytometry. For analysis of Ig secretion, the same V-transfection method as above was used. H The gene was cloned into the pFUSE vector containing the mouse IgG2a Fc. 293T / 17 cells were co-transfected with one of the HC and LC constructs, as described above, plus a human CD4 (hCD4) expression vector as a transfection control. Approximately 48 hours later, transfected cells and their corresponding supernatants were harvested and analyzed for HC / LC expression / secretion by Western blotting.

[0336] Collectively, the data from these experiments demonstrate that bovine VH domains containing either ultralong or normal-length HCDR3s and fused to mouse IgM backbones are highly sensitive to mismatched C L V with region κ or V λ , i.e., V λ and C κ or V κ and C λ When co-expressed with LCs bearing the same bovine V, cell surface IgM expression, as assessed by flow cytometry, was significantly higher than that of the control LCs bearing the same bovine V. H Match V L / C L LC with V λ C λ1 , C λ2 or C λ3 and bond or V κ C κ These results were confirmed by staining for cell surface CD79b, an obligate component of the BCR. The extent of the loss of expression was dependent on the specific V H Gene dependent; certain V HThe genes allowed some cell surface expression of hybrid light chains, but others were more restrictive. The same trend was observed for Ig secretion.

[0337] Figure 15 shows the mouse (m) constant region C κ , C λ1 , C λ2 or C λ3 Bovine (b) V bound to λ (AF023843.1) or V κ Figure 1 shows the results of flow cytometry analysis of cells expressing bovine BLV1H12, a VH domain with an ultralong HCDR3 and a mouse IgM backbone paired with (BC122795). The identity of each column in the flow cytometry profile is: 1501, bV κ / mC κ ;1502, bV κ / mC λ1 ;1503, bV κ / mC λ2 ;1504, bV κ / mC λ3 ;1505, bV λ / mC κ ;1506, bV λ / mC λ1 ;1507, bV λ / mC λ2 ;1508, bV λ / mC λ3 The panel in row 1509 was stained with hCD4, row 1510 with mouse IgM. bRow 1511 was stained with λLC, row 1512 was stained with κLC, and row 1513 was stained with CD79b, which are transfection controls. The x-axis (1514) of row 1 in all panels represents cell number. The x-axis of the remaining rows (1515-1518) in all panels represents % max. The frequency of untransfected hCD4+ cells is indicated by the number at the top left of each panel in row 1514, and the frequency of transfected hCD4+ cells is indicated by the number at the top right of each panel in row 1514. Transfection efficiency was similar in all cases. Differently shaded histograms in rows 1515-1518 in all panels show negative (1519) and positive (1520) staining with the specific antibody used in each row, gated on transfected hCD4+ cells (shown as examples in rows 1501 and 1515). The numbers at the top right in row 1515 of each panel are a quantitative indicator of the expression level of cell surface IgM on transfected, i.e., hCD4+ cells. b The mean fluorescence intensity (MFI) of the staining is shown. κ Mouse C λ1 , C λ1 or C λ2 (row 1515, panels 1502-1504), cell surface IgM expression was significantly higher than that of the same bovine V κ Mouse C κ (row 1515, panel 1501, MFI 7547) were lower than those bound to V (MFI 586, 449, 180, respectively). λ Bovine IgM with C κ (Row 1515, Panel 1505, MFI 6726) C λ1 , C λ1 or C λ2 (row 1515, panels 1506-1508, MFI 8017, 8951, 10783, respectively), although the difference in expression was not as dramatic in these cases.

[0338] Figure 16 shows the mouse (m) constant region C κ , C λ1 , C λ2 or C λ3Bovine (b) V bound to λ (AF023843.1) or V κ

[0039] Figure 1 shows the results of flow cytometry analysis of cells expressing bovine IGHV B4, a VH domain with a normal-length HCDR3 and a mouse IgM backbone paired with (BC122795). The identity of each row in the flow cytometry profile is: 1601, bV κ / mC κ ;1602, bV κ / mC λ1 ;1603, bV κ / mC λ2 ;1604, bV κ / mC λ3 ;1605, bV λ / mC κ ;1606, bV λ / mC λ1 ;1607, bV λ / mC λ2 ;1608, bV λ / mC λ3 The panel in row (1609) was stained with hCD4, and the panel in row (1610) was stained with mouse IgM. b Rows 1615-1618 in all panels are transfection controls stained with allotypes, row 1611 with λLC, row 1612 with κLC, and row 1613 with CD79b. The x-axis of row 1 (1614) in all panels indicates cell count. The x-axis of the remaining rows (1615-1618) in all panels indicates % max. The frequency of untransfected hCD4+ cells is indicated by the number at the top left of each panel in row 1614, and the frequency of transfected hCD4+ cells is indicated by the number at the top right of each panel in row 1614. Transfection efficiency was similar in all cases. Differently shaded histograms in rows 1615-1618 in all panels indicate negative (1619) and positive (1620) staining with the specific antibody used in each row gated on transfected hCD4+ cells (shown as examples in rows 1601 and 1615). The numbers at the top right in row 1615 of each panel are a quantitative indicator of the expression level of cell surface IgM on transfected, i.e., hCD4+ cells. b The mean fluorescence intensity (MFI) of the staining is shown.κ Mouse C λ1 , C λ1 or C λ2 (row 1615, panels 1602-1604), cell surface IgM expression was significantly higher than that of the same bovine V κ Mouse C κ (row 1615, panel 1601, MFI 6339) were lower than those bound to V (MFI 496, 566, 168, respectively). λ Bovine IgM with C κ (Row 1615, Panel 1605, 9103) C λ2 or C λ3 (row 1615, panels 1607 and 1608, MFI 10246 and 17240, respectively). However, in this case, V λ -C κ (9108)'s MFI is V λ -C λ1 It was actually higher than (7263).

[0339] The results of this analysis showed that bovine V λ and mouse C κ or Bovine V κ and mouse C λ1 , C λ2 or C λ3 We showed that hybrid light chains consisting of bovine V are relatively poorly expressed on the surface of cells bearing μHC. Because B cell survival depends on IgM BCR expression, bovine V λ and Mouse C κ It is clear that pairing with bovine V results in a significant decrease in the development of λLC-expressing B cells. κ and Mouse C λ1 , C λ2 or C λ3 Pairing of κ-LC reduces the outgrowth of κ-LC-expressing B cells.

[0340] Expression and secretion of Ig with hybrid or homologous LC. Supernatants and cell lysates from transiently transfected cells were analyzed by Western blotting. Figure 17 shows the expression and secretion of Ig with hybrid or homologous LC. κ , C λ1, C λ2 or C λ3 Bovine (b) V bound to λ (AF023843.1) or V κ Shown are the supernatants (1701) and cell lysates (1702) of cells expressing bovine BLV1H12, a VH domain with an ultralong HCDR3 and a mouse IgG2a HC backbone paired with (BC122795). Blots were electrophoresed under reducing conditions and probed with an antibody against mouse γ2a HC. Contents of each lane: 1703, bV κ / mC κ ;1704, bV κ / mC λ1 ;1705, bV κ / mC λ2 ;1706, bV κ / mC λ3 ;1707, bV λ / mC κ ;1708, bV λ / mC λ1 ;1709, bV λ / mC λ2 ;1710, bV λ / mC λ3

[0341] Molecular weight standards are in lane 1711. This particular bV κ / MC L The combination was not particularly well secreted regardless of LC, but clearly bV κ / mC κ (1703) is bV κ / mC λ2 (1705) or bV κ / mC λ3 (1706) was secreted better than bV. λ is mC κWhile (1707) was poorly secreted, all three mouse Cλ subclasses (1708-1710) were well secreted. Intracellular HC levels were similar for all eight constructs (blot 1702, lanes 1703-1710), indicating that differences in intracellular protein stability are responsible for the observed differences in secretion. Figure 18 shows loading controls using Myc (1802) and GAPDH (1803). Lane designations in this figure correspond to Figure 17.

[0342] Figure 19 shows the mouse (m) constant region C κ , C λ1 , C λ2 or C λ3 Bovine (b) V bound to λ (AF023843.1) or V κ Shown are the supernatants (1901) and cell lysates (1902) of cells expressing bovine BLV1H12, a VH domain with an average-length HCDR3 and a mouse IgG2a HC backbone paired with (BC122795). In this case, the blot was electrophoresed under non-reducing conditions and probed with an antibody against mouse γ2a HC. The contents of each lane are: 1903, bV κ / mC κ ;1904, bV κ / mC λ1 ;1905, bV κ / mC λ2 ;1906, bV κ / mC λ3 ;1907, bV λ / mC κ ;1908, bV λ / mC λ1 ;1909, bV λ / mC λ2 ;1910, bV λ / mC λ3 Molecular weight standards are in lane 1911. This particular bV κ / MC L The combination was not particularly well secreted regardless of LC, but clearly bV κ / mC κ (1903) is bV κ / mC λ2 (1905) or bV κ / mC λ3 (1906) was secreted better than bV. λ (1907) is mC κ While only very little was secreted from the constructs in Figure 19, all three mouse Cλ subclasses (1908-1910) were well secreted. Intracellular HC levels were similar for all eight constructs (blot 1902, lanes 1903-1910), indicating that differences in intracellular protein stability are responsible for the observed differences in secretion. Figure 20 shows loading controls using Myc (2001) and GAPDH (2002). Lane designations in this figure correspond to Figure 19.

[0343] Example 10: Expression of partially bovine immunoglobulins with mouse IgD IgD is co-expressed with IgM in mature B cells in most mammals. However, the question of whether cattle possess functional constant region genes encoding the δHC is quite controversial. Previous studies found no evidence of the Cδ gene or IgD protein in cattle (Butler, et al. ibid; Naessens J ibid). Subsequently, evidence for the existence of the bovine Cδ gene (Zhao, et al. ibid) and the expression of bovine IgD (Xu, et al. ibid) was reported, but the presence of bovine IgD was not fully documented. + The reported frequency of IGH cells was much lower than in mice. The current annotation of the bovine IGH locus by the International ImMunoGeneTics information system® / www.imgt.org (IMGT) describes four Cδ genes, IGHDD1P, IGHDD2P, IGHDD39, and IGHD, none of which are functional. There are two IGHD alleles, one of which is a pseudogene due to a frameshift in M1, and the other is an ORF due to a non-canonical splice donor in NGC rather than NGT. The IgD observed by Xu et al. + The low frequency of cells is probably due to occasional leaky splicing from the ORF allele. H Partially or completely non-functional gene regions δBecause they appear to have evolved with the gene, cow V H The concern is that the domain may not fold properly when bound to mouse Cδ, and partial or absent assembly of bovine IgD may interfere with normal B cell development.

[0344] Bovine V with a Cδ backbone H To test whether the domains could be assembled into IgD molecules that could be expressed on the cell membrane, transient transfection and flow cytometry analysis was performed using methods similar to those described in Example 9.

[0345] 293T / 17 cells were co-transfected with the HC constructs from Example 9 (IGHV BLV1H12 or B4), except that Cμ was replaced with Cδ, as well as an additional IGHV BL5B8 linked to Cδ, along with a human CD4 (hCD4) expression vector as a transfection control. Both IGHVs BLV1H12 and B4 have ultralong HCDR3 regions of 61 and 56 amino acids, respectively. Each heavy chain construct was co-transfected with one of the λLC constructs described in Example 9 along with the CD79a / b expression vector. As can be seen in Figures 21-24, the HC with a bovine VH domain with a murine IgD backbone was expressed as a bovine VH domain. λ -Mouse C λ1 When paired with the mouse IgD backbone and bovine VLC, they are expressed intracellularly and on the cell surface. λ -Mouse C λ1 (2103), C λ2 (2104) or C λ3These figures show the expression of intracellular bovine IGHV BLV1H12 (2101) and bovine IGHV BLV5B8 (2102) with IgG (2105). In these studies, row 2106 shows staining for intracellular hCD4, a control for transfection efficiency. The frequency of untransfected hCD4 cells is indicated by the numbers at the bottom left of each panel in row 2106, and the frequency of transfected hCD4+ cells is indicated by the numbers at the bottom right of each panel in row 2106. Transfection efficiency was similar in all cases. Differently shaded histograms in rows 2107-2109 in all panels show negative (2110) and positive (2111) staining with the specific antibody used in each row, gated on transfected hCD4+ cells (shown as examples in rows 2103 and 2107). Row 2107 shows staining for intracellular IgD, row 2108 shows staining for intracellular λLC, and row 2109 shows staining for intracellular CD79b. H -mC δ / bV λ -mC λ The combinations were well expressed intracellularly in transfected cells. Figure 22 shows cell surface expression of the same bovine constructs as in Figure 21 stained with the same antibodies. All were well expressed at the cell surface based on IgD, λLC, or CD79b staining. (Cell surface staining data is in the same layout as Figure 21.) Figure 23 shows the expression of mouse C λ1 (2302), C λ2 (2303) or C λ3These figures show the expression of intracellular bovine IGHV B4 with a mouse IgD backbone and an average-length HCDR3 with a bovine Vλ (2304). In these studies, row 2305 shows staining of intracellular hCD4, a control for transfection efficiency. The frequency of untransfected hCD4 cells is indicated by the numbers at the bottom left of each panel in row 2305, and the frequency of transfected hCD4+ cells is indicated by the numbers at the bottom right of each panel in row 2305. Transfection efficiency was similar in all cases. Differently shaded histograms in rows 2306-2308 in all panels show negative (2309) and positive (2310) staining with the specific antibody used in each row, gated on transfected hCD4+ cells (shown as examples in rows 2302 and 2306). Row 2306 shows staining for intracellular IgD, row 2307 shows staining for intracellular λLC, and row 2308 shows staining for intracellular CD79b in these particular bovine bV H -mC δ / bV λ -mC λ The combination was successfully expressed intracellularly in transfected cells.

[0346] Figure 24 also shows mouse C λ1 (2402), C λ2 (2403) or C λ3 This figure shows cell surface staining of expressed bovine IGHV B4 with a mouse IgD backbone and bovine Vλ bound to (2404). (The cell surface staining data are in the same layout as in Figure 23.) These specific bovine bV H -mC δ / bV λ -mC λ The combination was successfully expressed on the surface of transfected cells.

[0347] Therefore, all three tested bovine V H The domains were expressed on a murine IgD backbone, but the level of cell surface expression varied somewhat depending on the particular HC / LC combination. HC / LC combinations that can be expressed as IgD on the cell surface are thought to be secreted into the follicular B cell compartment during B cell development and generate the appropriate BCR repertoire.

[0348] The foregoing merely illustrates the principles of the methods described herein. It will be recognized that those skilled in the art will be able to devise various adaptations, not explicitly described or depicted herein, which embody the principles of the present invention and are encompassed within its spirit and scope. Furthermore, all exemplary and conditional language described herein is intended primarily to aid the reader in understanding the principles of the present invention and the concepts intended by the inventors to advance the art, and should not be construed as being limited to such specifically described examples and conditions. Furthermore, all statements herein describing principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass structural and functional equivalents thereof. Furthermore, such equivalents are intended to include both currently known equivalents and future-developed equivalents, i.e., any elements under development, that perform the same function, regardless of structure. Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention are embodied by the appended claims. In the following claims, unless the term "means" is used, no feature or element described therein is to be construed as a means-plus-function limitation pursuant to 35 U.S.C. 112, paragraph 6. All references cited herein are incorporated by reference in their entirety for all purposes.

[0349] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] Table 7 Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 Table 20 Table 21 Table 22 Table 23 Table 24 Table 25 Table 26 Table 27 Table 28 Table 29 Table 30 Table 31 Table 32 Table 33 Table 34 Table 35 Table 36 Table 37

Claims

1. A transgenic rodent or rodent cell comprising an engineered partially bovine immunoglobulin light chain locus comprising a bovine immunoglobulin lambda light chain variable region gene segment, wherein the engineered immunoglobulin locus is capable of expressing immunoglobulins comprising bovine variable domains, and wherein the transgenic rodent produces or is more likely to produce immunoglobulins comprising lambda light chains than immunoglobulins comprising kappa light chains.

2. 2. The transgenic rodent of claim 1, wherein more lambda light chain producing cells are likely to be isolated from said rodent than kappa light chain producing cells.

3. 2. The transgenic rodent of claim 1, wherein the transgenic rodent produces immunoglobulins comprising at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100% λ light chains.

4. 2. The transgenic rodent cell of claim 1, wherein the probability that the transgenic rodent cell or its progeny will produce immunoglobulins comprising a λ light chain is at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100%.

5. The engineered immunoglobulin locus is bovine V λ Gene segment coding sequence and J λ 5. The transgenic rodent or rodent cell of any of claims 1-4, comprising gene segment coding sequences and rodent immunoglobulin light chain variable region gene locus rodent non-coding regulatory sequences or scaffold sequences.

6. A bovine V gene in which an engineered immunoglobulin locus is embedded in rodent non-coding regulatory sequences or scaffold sequences of a rodent immunoglobulin lambda light chain variable region gene locus. λ and J. λ 6. The transgenic rodent or rodent cell of any of claims 1 to 5, comprising a gene segment coding sequence.

7. Partially bovine immunoglobulin loci at one or more bovine V λ and J. λ 7. The transgenic rodent or rodent cell of any of claims 1 to 6, comprising a gene segment coding sequence and one or more rodent immunoglobulin lambda constant region coding sequences.

8. A bovine V gene in which an engineered immunoglobulin locus is embedded in a rodent non-coding regulatory sequence or scaffold sequence of a rodent immunoglobulin kappa light chain variable region gene locus. λ and J. λ 5. The transgenic rodent or rodent cell of any of claims 1 to 4, comprising a gene segment coding sequence.

9. Bovine V having one or more engineered immunoglobulin variable region loci λ a gene segment coding sequence and one or more J-C units, wherein each J-C unit is a bovine J λ 9. The transgenic rodent or rodent cell of claim 8, comprising a gene segment coding sequence and a rodent lambda constant region coding sequence.

10. The rodent lambda constant region coding sequence is a rodent C λ１ , C λ２ , C λ３ 10. The transgenic rodent or rodent cell of claim 9, comprising a coding sequence or a combination thereof.

11. one or more bovine V located upstream of one or more J-C units λ gene segment coding sequences, wherein each J-C unit is a bovine J λ Gene segment coding sequences and rodent C λ 11. The transgenic rodent or rodent cell of claims 9-10, comprising a gene segment coding sequence.

12. one or more bovine V located upstream of one or more J-C units λ gene segment coding sequences, wherein each J-C unit is a bovine J λ Gene segment coding sequences and rodent C λ Gene segment coding sequences and rodent C λ 11. The transgenic rodent or rodent cell of claim 9 or 10, comprising a non-coding sequence.

13. A bovine J-C unit embedded in a non-coding regulatory or scaffold sequence of the rodent immunoglobulin kappa light chain locus. λ 13. The transgenic rodent or rodent cell of any of claims 9 to 12, comprising a gene segment coding sequence and a rodent lambda constant region coding sequence.

14. The engineered immunoglobulin locus comprises a rodent immunoglobulin κ locus, wherein one or more rodent V κ gene segment coding sequences and one or more rodent J κ The gene segment coding sequence is deleted, and each of the gene segments is one or more bovine V λ A gene segment coding sequence and one or more J λ The gene segment coding sequence is replaced by a rodent C at the locus. κ The coding sequence is rodent C λ１ , C λ２ , C λ３ 9. The transgenic rodent or rodent cell of claim 8, wherein the gene encoding the gene for the transgenic rodent is replaced with a coding sequence or a combination thereof.

15. A rodent C with one or more engineered immunoglobulin loci λ One or more bovine J genes upstream of the coding sequence λ One or more bovine V sequences upstream of the gene segment coding sequence λ 15. The transgenic rodent or rodent cell of claim 14, comprising a gene segment coding sequence.

16. The endogenous rodent immunoglobulin kappa light chain locus is deleted, inactivated, or a. All endogenous rodent V κ deletions or mutations in gene segment coding sequences; b. All endogenous rodent J κ deletions or mutations in gene segment coding sequences; c. All endogenous rodent C κ Deletions or mutations in coding sequences; d. Rodent C κ deletions or mutations of the 5′ splice site and adjacent polypyrimidine tract of the coding sequence; e. Endogenous intron kappa enhancer (iE κ ) and deletion, mutation, or disruption of the 3' enhancer sequence 16. The transgenic rodent or rodent cell of any of claims 1 to 15, which is rendered non-functional by one or more of the following:

17. Expression of endogenous rodent immunoglobulin λ light chain variable domains a. All endogenous rodent V λ deletions or mutations of gene segments; b. All endogenous rodent J λ deletions or mutations of gene segments; and c. All endogenous rodent C λ Deletions or mutations in the coding sequence 17. The transgenic rodent or rodent cell of any of claims 1 to 16, wherein the transgenic rodent or rodent cell is repressed or inactivated by one or more of the following:

18. 18. The transgenic rodent or rodent cell of any of claims 1 to 17, wherein the engineered immunoglobulin locus expresses an immunoglobulin light chain comprising a bovine λ variable domain and a rodent λ constant domain.

19. The genome of the transgenic rodent or rodent cell contains bovine V κ and J. κ 5. The transgenic rodent or rodent cell of any of claims 1 to 4, comprising an engineered immunoglobulin locus comprising a gene segment coding sequence.

20. Cow V κ and J. κ 20. The transgenic rodent or rodent cell of claim 19, wherein the gene segment coding sequence is inserted into a rodent immunoglobulin kappa light chain locus.

21. Cow V κ and J. κ 21. The transgenic rodent or rodent cell of claim 19 or 20, wherein the gene segment coding sequence is embedded in non-coding regulatory or scaffolding sequences of a rodent immunoglobulin kappa light chain variable region gene locus.

22. Cow V κ and J. κ 22. The transgenic rodent or rodent cell of any of claims 19 to 21, wherein the coding sequence is inserted upstream of a rodent immunoglobulin kappa light chain constant region coding sequence.

23. The genome of a transgenic rodent or rodent cell contains a bovine V gene inserted into the rodent immunoglobulin lambda light chain locus. κ and J. κ 5. The transgenic rodent or rodent cell of any of claims 1 to 4, comprising an engineered immunoglobulin locus comprising a gene segment coding sequence.

24. Cow V κ and J. κ 24. The transgenic rodent or rodent cell of claim 23, wherein the gene segment coding sequence is embedded in rodent non-coding regulatory or scaffolding sequences of a rodent immunoglobulin lambda light chain variable region gene locus.

25. Cow V κ and J. κ 25. The transgenic rodent or rodent cell of claim 23 or 24, comprising a rodent immunoglobulin kappa light chain constant region coding sequence inserted downstream of the gene segment coding sequence.

26. The rodent immunoglobulin κ light chain constant region is endogenous rodent C λ２ 26. The transgenic rodent or rodent cell of claim 25, wherein the gene is inserted upstream of a coding sequence.

27. Expression of endogenous rodent immunoglobulin λ light chain variable domains a. All endogenous rodent V λ deletions or mutations in gene segment coding sequences; b. All endogenous rodent J λ Deletions or mutations in gene segment coding sequences; and c. Total endogenous C λ Deletions or mutations in coding sequences or splice sites 27. The transgenic rodent or rodent cell of any of claims 23 to 26, wherein the transgenic rodent or rodent cell is repressed or inactivated by one or more of:

28. The engineered bovine immunoglobulin light chain locus integrates the rodent intronic kappa enhancer (iE κ ) and 3′E κ 28. The transgenic rodent or rodent cell of any of claims 1 to 27, comprising a regulatory sequence.

29. 29. The transgenic rodent or rodent cell of any of claims 1 to 28, wherein the transgenic rodent or rodent cell comprises an engineered partially bovine immunoglobulin heavy chain locus comprising bovine immunoglobulin heavy chain variable region gene coding sequences and non-coding regulatory or scaffolding sequences of the rodent immunoglobulin heavy chain locus.

30. The engineered bovine immunoglobulin heavy chain locus is bovine V Ｈ , D and J Ｈ 30. The transgenic rodent or rodent cell of claim 29 comprising the gene segment.

31. Each cow V Ｈ , D or J Ｈ A V coding gene segment embedded in non-coding regulatory or scaffolding sequences of the rodent immunoglobulin heavy chain locus. Ｈ , D or J Ｈ 31. The transgenic rodent or rodent cell of claim 30 comprising a coding sequence.

32. 32. The transgenic rodent or rodent cell of claim 31 , wherein heavy chain rodent non-coding regulatory or scaffolding sequences are interspersed with functional ADAM6A genes, ADAM6B genes, or a combination thereof.

33. 33. The transgenic rodent or rodent cell of any of claims 1 to 32, wherein the rodent regulatory or scaffolding sequences comprise enhancers, promoters, splice sites, introns, recombination signal sequences, or combinations thereof.

34. 34. The transgenic rodent or rodent cell of any of claims 1 to 33, wherein endogenous rodent immunoglobulin loci have been deleted and replaced with engineered partial bovine immunoglobulin loci.

35. 35. The transgenic rodent or rodent cell of any of claims 1 to 34, wherein the rodent is a mouse or a rat.

36. 36. The transgenic rodent or rodent cell of any of claims 1 to 35, wherein the rodent cell is an embryonic stem (ES) cell or a cell of an early embryo.

37. 37. The transgenic rodent or rodent cell of any of claims 1 to 36, wherein the rodent cell is a mouse or rat embryonic stem (ES) cell or an early embryonic mouse or rat cell.

38. 38. A cell of the B lymphocyte lineage obtained from the transgenic rodent of any of claims 1 to 37, wherein the engineered immunoglobulin locus expresses a chimeric immunoglobulin heavy or light chain comprising a bovine variable region and a rodent immunoglobulin constant region.

39. A hybridoma cell or immortalized cell line derived from a cell of the B lymphocyte lineage of claim 38.

40. 40. An antibody or antigen-binding portion thereof produced by the cell of claim 38 or 39.

41. V from the immunoglobulin produced by the cell of claim 38 or 39. Ｈ , D or J Ｈ or V Ｌ or J Ｌ Nucleic acid sequences of gene segment coding sequences.

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