Genetically modified mouse for preparing antibody and preparation method therefor

Abstract

Disclosed in the present invention is a genetically modified mouse in which the immunoglobulin locus is engineered to be inserted with a gene segment of a human immunoglobulin variable region. The mouse is capable of reproducing normally and producing a human-mouse chimeric antibody, wherein the human-mouse chimeric antibody contains a human variable region and a mouse constant region. Further provided in the present invention is a method for preparing the genetically modified mouse and the use of the mouse.

Description

Genetically modified mice for preparing antibodies and preparation method thereof Technical Field

[0001] The present invention relates to genetically modified mice, cells, embryos, and tissues. Specifically, the present invention relates to mice in which both the immunoglobulin heavy chain variable region genome and the kappa light chain variable region genome have been humanized, and methods for preparing the same. The present invention also relates to the genome of the modified mice, cells and tissues containing the genome, and methods and uses of monoclonal antibodies prepared using the mice. The present invention also relates to mice having the modified genome. Background Art

[0002] Humanized mice show the humoral immune system with full function, and it is basically no different from the humoral immune system of wild-type mice.They show normal cell colony in all stages of B cell development.They show normal lymphoid organ morphology.The antibody sequence of mouse shows normal V (D) J rearrangement and normal somatic hypermutation frequency.The antibody colony in these mice reflects the isotype distribution caused by normal species type conversion (such as normal isotype cis conversion).Mouse is carried out immunity and caused powerful humoral immune response, and it produces the huge and diverse antibody pedigree with human immunoglobulin variable region that is suitable for being used as treatment candidate.

[0003] With human immunoglobulin variable sequence, mouse immunoglobulin variable sequence is accurately replaced, so that genetic modification or transgenic mouse can be formed. However, because of the divergent evolution of immunoglobulin locus between mouse and human, even if by carrying out continuous recombineering to very large human immunoglobulin sequence chain segment, thus accurately replacing heavy chain and light chain locus with corresponding human immunoglobulin sequence, some problems may also occur. For example, the intergenic sequence interspersed in immunoglobulin locus is inconsistent between mouse and human, and may not be functionally equivalent in some cases. The difference between mouse and human on its immunoglobulin locus still may cause the abnormality of humanized mouse, especially when some parts of endogenous mouse immunoglobulin heavy chain locus are humanized or other manipulations are carried out. Some modifications at mouse immunoglobulin heavy chain locus are harmful, such as the loss of the ability of modified mouse to mate and produce offspring. It has been found that the decline or disappearance of the fertility of male mice is relevant to the impairment of Adam6 gene.

[0004] ADAM6 protein is a member of the ADAM protein family, where ADAM is the acronym for A disintegrin and metalloprotease. The ADAM protein family is large and diverse and has multiple functions including cell adhesion. Some members of the ADAM family are involved in spermatogenesis and fertilization. For example, ADAM2 encodes a subunit of the protein fertilin involved in sperm-egg interaction. ADAM3 or cyritestin appears to be necessary for the binding of sperm to the zona pellucida. The absence of ADAM2 or ADAM3 will lead to infertility. It has been hypothesized that ADAM2, ADAM3 and ADAM6 form a complex on the surface of mouse sperm cells. The human ADAM6 gene is located between human VH genes VH1-2 and VH6-1. In mice, there are two ADAM6 genes, ADAM6a and ADAM6b, present in the intergenic region between the mouse VH and DH gene segments, and in mice, the transcription direction of the ADAM6a and ADAM6b genes is opposite to the transcription direction of the surrounding immunoglobulin gene segments.

[0005] CN105861548B discloses a kind of ADAM6 mouse, wherein the immunoglobulin heavy chain gene seat in the germline of the mouse inserts one or more human immunoglobulin gene sequences, wherein the insertion destroys the function of endogenous ADAM6 gene;Then, the nucleotide sequence encoding mouse ADAM6a protein and the nucleotide sequence encoding mouse ADAM6b protein are inserted in the germline of the mouse, wherein the mouse ADAM6a protein and the mouse ADAM6b protein are expressed by the nucleotide sequence, wherein the mouse ADAM6a protein and the mouse ADAM6b protein are expressed from the nucleotide sequence and improve or restore fertility when expressed in male mice. The destruction of endogenous ADAM6 gene is undesirable, and it may have an impact on the long-term reproductive capacity of mice.

[0006] In view of this, the present invention provides a mouse genome with a modified heavy chain locus, which retains the endogenous ADAM6 gene and its function, and is capable of producing human-mouse chimeric antibodies while maintaining fertility.

[0007] Summary of the Invention

[0008] One aspect of the present invention provides a method for preparing a genetically modified mouse, the method comprising:

[0009] (i) obtaining a heavy chain gene locus modified mouse, comprising

[0010] (ia) inserting a first partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the first mouse, wherein the first partial segment includes a first partial hIgHV continuous segment, the entire hIgHD segment, and the entire hIgHJ segment, the first partial segment does not include a segment between the downstream of the hIgHV1-2 gene and the upstream of the hIgHV6-1 gene, and includes a first recombination site upstream of the first partial segment and downstream of the mIgHJ region;

[0011] (ib) inserting a second partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of a second mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment includes a second partial hIgHV continuous segment, and includes a second recombination site between the downstream of the second partial hIgHV continuous segment and the upstream of the mIgHC region;

[0012] (ic) hybridizing the first mouse with the second mouse, and screening to obtain a third mouse, wherein the second partial segment of the human immunoglobulin heavy chain variable region locus and the first partial segment located downstream of the second partial segment are inserted between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the third mouse, and a third recombination site is present between the second partial segment and the first partial segment; and

[0013] (id) knocking out the mIgHV5-1 gene and all upstream mIgHV segments of the immunoglobulin heavy chain locus, the continuous segment between the mIgHD1-1 gene and the mIgHJ4 gene, and the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes of the third mouse, respectively, to obtain the heavy chain locus gene-modified mice;

[0014] (ii) obtaining a Kappa light chain gene locus modified mouse, wherein the immunoglobulin Kappa light chain gene locus of the Kappa light chain modified mouse includes all hIgKV segments, all hIgKJ segments, and mIgKC segments in sequence, and all mIgKV segments and all mIgKJ segments are knocked out; and

[0015] (iii) breeding the heavy chain locus modified mice obtained in step (i) and the kappa light chain locus modified mice obtained in step (ii) to screen for double-positive mice, and interbreeding the obtained double-positive mice to screen for homozygous mice.

[0016] In the present invention, the first part of the hIgHV continuous segment of the first part of the human immunoglobulin heavy chain variable region locus can start from any hIgHV segment gene upstream of the hIgHV1-2 gene (including functional genes, pseudogenes or ORFs) and end at the hIgHV1-2 gene.

[0017] In one embodiment, the gene of any hIgHV segment upstream of the hIgHV1-2 gene can be any functional gene, pseudogene or ORF between hIgHV(III)-82 and hIgHV1-2 genes. In one embodiment, the gene of any hIgHV segment upstream of the hIgHV1-2 gene is a functional gene, such as hIgHV3-74, hIgHV3-73, hIgHV3-72, hIgHV2-70, hIgHV1-69D, hIgHV1-69-2, hIgHV2-70D, hIgHV1-69, hIgHV3-66, hIgHV3-64 , hIgHV3-62, hIgHV4-61, hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgH V3-48, hIgHV1-46, hIgHV1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3- 35. hIgHV4-34, hIgHV3-33, hIgHV4-31, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4 -30-2, hIgHV4-30-1, hIgHV3-30, hIgHV4-28, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3- 23. hIgHV3-21, hIgHV3-20, hIgHV1-18, hIgHV3-15, hIgHV3-13, hIgHV3-11, hIgHV5-10-1, hIgHV3-9, hIgHV3-64D, hIgHV3-8, hIgHV3-7, hIgHV2-5, hIgHV7-4-1, hIgHV4-4 or hIgHV1-3.

[0018] In one embodiment, the first portion of the first segment of the continuous segment of hlgHV starts with hlgHV3-74, hlgHV3-73, hlgHV3-72, hlgHV2-70, hlgHV1-69D, hlgHV1-69-2, hlgHV2-70D, hlgHV1-69, hlgHV3-66, hlgHV3-64, hlgHV3-62, hlgHV4- 61. hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgHV3-48, hIgHV1-46, hIgHV 1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3-35, hIgHV4-34, hIgHV3-33, hIgHV4-31, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4-30-2, hIgHV4-30-1, hIgHV3- 30. hIgHV4-28, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3-23, hIgHV3-21, hIgHV3-20, hIgH V1-18, hIgHV3-15, hIgHV3-13, hIgHV3-11, hIgHV5-10-1, hIgHV3-9, hIgHV3-64D, hIgHV3-8, hIgHV3-7, hIgHV2-5, hIgHV7-4-1, hIgHV4-4 or hIgHV1-3, terminated at the hIgHV1-2 gene, all comprising the described genes as ends. In one embodiment, the first portion of the first segment is a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0019] In the present invention, the second portion of the heavy chain variable region genome is located upstream of the first portion of the heavy chain variable region genome, and the second portion of the hIgHV continuous segment can start at any gene (including functional genes, pseudogenes, or ORFs) of the hIgHV segment and end at any gene (including functional genes, pseudogenes, or ORFs) of the hIgHV segment upstream of the starting gene of the first portion of the hIgHV continuous segment. In some embodiments, the second portion of the segment and the first portion of the segment together constitute all functional genes upstream of hIgHV1-2 and the continuous segment therebetween, i.e., the continuous segment between the hIgHV3-74 and hIgHV1-2 genes. For example, in some embodiments, the second part of the hIgHV continuous segment includes or is a continuous segment between the hIgHV3-74 gene and the hIgHV3-30 gene, and the first part of the hIgHV continuous segment includes or is a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0020] In some embodiments, the immunoglobulin heavy chain locus of the obtained genetically modified mouse includes, in sequence: (i) mouse Adam6a gene; (ii) mouse Adam6b gene; (iii) a continuous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (iv) a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (v) a continuous segment between the hIgHV6-1 gene and the hIgHJ6 gene; and (vi) the mIgHC region.

[0021] In some embodiments, in step (ia), the first partial hIgHV continuous segment, the entire hIgHD segment, and the entire hIgHJ segment of the first partial segment are inserted between the mIgHJ region and the mIgHC region by at least two steps, and the at least two steps include:

[0022] (ia1) inserting the first continuous segment comprising hIgHV6-1 to hIgHJ6 genes between the mIgHJ region and the mIgHC region; and

[0023] (ia2) inserting a second continuous segment comprising hIgHV4-28 to hIgHV1-2 genes upstream of the first continuous segment.

[0024] In some embodiments, step (ia1) specifically includes:

[0025] (ia11) inserting a continuous segment comprising hIgHD6-25 to hIgHJ6 genes between the mIgHJ region and the mIgHC region;

[0026] (ia12) inserting a continuous segment comprising hIgHD6-13 to hIgHD5-24 genes upstream of the inserted segment in step (ia11);

[0027] (ia13) inserting a continuous segment comprising hIgHD1-1 to hIgHD5-12 genes upstream of the inserted segment in step (ia12); and

[0028] (ia14) Insert the continuous segment between hIgHV6-1 and hIgHD1-1 genes and loxP and lox2272-PB5' sites upstream of the inserted segment in step (ia13).

[0029] In some embodiments, step (ia2) specifically includes:

[0030] (ia21) recombining the genome obtained in step (ia1) with a BAC vector comprising a second continuous segment between hIgHV4-28 and hIgHV1-2 genes, loxP and lox2272 sites located at both ends of the second continuous segment in the same orientation as in step (ia14), and Cre recombinase, wherein PB3' is contained between the lox2272 site and the second continuous segment; and

[0031] (ia22) contacting the recombinant genome obtained in step (ia21) with PiggyBac transposase to screen for a genome comprising the first partial segment and the loxP site located upstream of the first partial segment.

[0032] In some embodiments, in step (ib), the second partial hIgHV continuous segment of the second partial segment is inserted upstream of the first partial segment by at least the following steps, including:

[0033] (ib1) inserting a continuous segment comprising hIgHV3-74 to hIgHV3-72 genes between the mIgHJ region and the mIgHC region; and

[0034] (ib2) inserting the continuous segment between hIgHV2-70 and hIgHV1-69D genes and the PB3'-lox5171 and loxP sites downstream of the inserted segment in step (ib1);

[0035] (ib3) recombining the genome obtained in step (ib2) with a BAC vector comprising a continuous segment between hIgHV1-69-2 and hIgHV3-30 genes and loxP and lox5171 located at both ends in the same direction as in step (ib2), and Cre recombinase, wherein PB5' is contained between the lox5171 site and the continuous segment described in this step;

[0036] (ib4) contacting the recombinant genome obtained in step (ib3) with PiggyBac transposase to screen for a genome comprising the second partial segment and the loxP site located downstream of the second partial segment.

[0037] In some embodiments, step (ic) comprises:

[0038] (ic1) screening Cre-positive mice containing the first and second segments;

[0039] (ic2) mating the mice obtained in step (ic1) with wild-type mice, and screening mice that do not carry Cre but contain the first partial segment and the second partial segment, wherein there is a loxP site between the second partial segment and the first partial segment.

[0040] In some embodiments, step (id) comprises:

[0041] (id1) mating the third mouse with wild-type mice to screen positive mice;

[0042] (id2) The positive mice obtained in step (id1) are mated between males and females to screen for homozygous mice.

[0043] In some embodiments, step (ii) comprises:

[0044] (iia) inserting a first partial segment of a human immunoglobulin kappa light chain variable region locus between the mIgKJ region and the mIgKC region of the immunoglobulin kappa light chain locus of a fourth mouse, wherein the first partial segment includes a first partial hIgKV continuous segment and the entire hIgKJ segment, and includes a fourth recombination site upstream of the first partial segment and downstream of the mIgKJ region;

[0045] (iib) inserting a second partial segment of the human immunoglobulin Kappa light chain variable region locus between the mIgKJ region and the mIgKC region of the immunoglobulin Kappa light chain locus of the fifth mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment includes a second partial hIgKV continuous segment, the first partial hIgKV continuous segment and the second partial hIgKV continuous segment together constitute the entire hIgKV segment, and include a fifth recombination site between the downstream of the second partial hIgKV continuous segment and the upstream of the mIgKC region;

[0046] (iic) hybridizing the fourth mouse with the fifth mouse to obtain a sixth mouse through screening, wherein the second partial segment of the human immunoglobulin Kappa light chain variable region locus and the first partial segment located downstream of the second partial segment are inserted between the mIgKJ region and the mIgKC region of the immunoglobulin Kappa light chain locus of the sixth mouse, and the sixth mouse has a sixth recombination site between the second partial segment and the first partial segment;

[0047] (iid) Knock out all the mIgKV segments and all the mIgKJ segments of the immunoglobulin Kappa light chain locus of the sixth mouse to obtain the Kappa light chain locus modified mouse.

[0048] In the present invention, the first part of the hIgKV continuous segment of the first part segment of the human immunoglobulin Kappa light chain variable region locus can start from any hIgKV segment gene upstream of the hIgKV4-1 gene (including functional genes, pseudogenes or ORFs) and end at the hIgKV4-1 gene.

[0049] In one embodiment, the gene of any hIgKV segment upstream of the hIgKV4-1 gene can be any functional gene, pseudogene or ORF between the hIgKV3D-7 and hIgKV4-1 genes. In one embodiment, any gene of the hIgKV segment upstream of the hIgKV4-1 gene is a functional gene or ORF, such as hIgKV3D-7, hIgKV1D-8, hIgKV1D-43, hIgKV1D-42, hIgKV3D-11, hIgKV1D-12, hIgKV1D-13, hIgKV3D-15, hIgKV1D-16, hIgKV1D-17, hIgKV6D-41, hIgKV3D-20, hIgKV6D-21, hIgKV2D-24, hIgKV2D-26, hIgKV2D-28, hIgKV2D-29, hIgKV2D-30, hIgKV1D-33 , hIgKV1D-37, hIgKV1D-39, hIgKV2D-40, hIgKV2-40, hIgKV1-39, hIgKV1-37, hIgKV1-33, hIgKV2-30, hIgKV2-29, hIgKV2-28, hIgKV2-27, hIgKV2-24, hIgKV 6-21, hIgKV3-20, hIgKV1-17, hIgKV1-16, hIgKV3-15, hIgKV1-13, hIgKV1-12 , hIgKV3-11, hIgKV1-9, hIgKV1-8, hIgKV3-7, hIgKV1-6, hIgKV1-5 or hIgKV5-2.

[0050] In one embodiment, the first portion of the first segment of the continuous segment starts with hIgKV3D-7, hIgKV1D-8, hIgKV1D-43, hIgKV1D-42, hIgKV3D-11, hIgKV1D-12, hIgKV1D-13, hIgKV3D-15, hIgKV1D-16, hIgKV1D-17, hIgKV1D-18, hIgKV1D-20, hIgKV1D-21, hIgKV1D-22, hIgKV1D-23, hIgKV1D-24, hIgKV1D-25, hIgKV1D-26, hIgKV1D-27, hIgKV1D-28, hIgKV1D-29, hIgKV1D-30, hIgKV1D-31, hIgKV1D-32, hIgKV1D-33, hIgKV1D-34, hIgKV1D-35, hIgKV1D-36, hIgKV1D-37, hIgKV1D-38, hIgKV1D-39, hIgKV1D-40, hIgKV1D-41, hIgKV1D-42 IgKV1D-17, hIgKV6D-41, hIgKV3D-20, hIgKV6D-21, hIgKV2D-24, hIgKV2D-26, h IgKV2D-28, hIgKV2D-29, hIgKV2D-30, hIgKV1D-33, hIgKV1D-37, hIgKV1D-39, hI gKV2D-40, hIgKV2-40, hIgKV1-39, hIgKV1-37, hIgKV1-33, hIgKV2-30, hIgKV2- 29. hIgKV2-28, hIgKV2-27, hIgKV2-24, hIgKV6-21, hIgKV3-20, hIgKV1-17, hIg One of hIgKV1-16, hIgKV3-15, hIgKV1-13, hIgKV1-12, hIgKV3-11, hIgKV1-9, hIgKV1-8, hIgKV3-7, hIgKV1-6, hIgKV1-5, or hIgKV5-2, terminates at the hIgKV4-1 gene, and all contain the described genes as ends. In one embodiment, the first portion of the first segment is a continuous segment between the hIgKV1-37 gene and the hIgKV4-1 gene.

[0051] In the present invention, the second portion of the kappa light chain variable region genome is located upstream of the first portion of the kappa light chain variable region genome, and the second portion of the hIgKV continuous segment can start at any gene (including functional genes, pseudogenes, or ORFs) of the hIgKV segment and end at any gene (including functional genes, pseudogenes, or ORFs) of the hIgKV segment upstream of the starting gene of the first portion of the hIgKV continuous segment. In some embodiments, the second portion of the segment and the first portion of the segment together constitute all functional genes upstream of hIgKV4-1 and the continuous segment therebetween, i.e., the continuous segment between the hIgKV4-1 and hIgKV3D-7 genes. For example, in some embodiments, the second part of the hIgKV continuous segment includes or is a continuous segment between the hIgKV3D-7 gene and the hIgKV2D-40 gene, and the first part of the hIgKV continuous segment includes or is a continuous segment between the hIgKV1-37 gene and the hIgKV4-1 gene.

[0052] In some embodiments, in step (iia), the first portion of the hIgKV continuous segment and the entire hIgKJ segment of the first portion are inserted between the mIgKJ region and the mIgKC region by at least two steps, wherein the at least two steps comprise:

[0053] (iia1) inserting a first continuous segment comprising hIgKV1-5 to hIgKJ5 genes between the mIgKJ region and the mIgKC region; and

[0054] (iia2) inserting a second continuous segment comprising hIgKV1-37 to hIgKV1-6 genes upstream of the first continuous segment.

[0055] In some embodiments, the step (iia1) specifically comprises:

[0056] (iia11) inserting a continuous segment comprising hIgKJ1 to hIgKJ5 genes between the mIgKJ region and the mIgKC region;

[0057] (iia12) inserting a continuous segment comprising hIgKV4-1 to hIgKV5-2 genes upstream of the inserted segment in step (iia11);

[0058] (iia13) inserting a continuous segment gene comprising the upstream of hIgKV5-2 to the downstream of hIgHKV2-4 upstream of the insertion segment of step (iia12); and

[0059] (iia14) Insert the continuous segment between hIgHKV2-4 and hIgKV1-5 genes and loxP and lox2272-PB5' sites upstream of the inserted segment in step (iia13).

[0060] In some embodiments, the step (iia2) specifically comprises:

[0061] (iia21) recombining the genome obtained in step (iia1) with a BAC vector comprising a second continuous segment between hIgKV1-6 and hIgKV1-37 genes, loxP and lox2272 sites located at both ends of the second continuous segment in the same orientation as in step (iia14), and Cre recombinase, wherein PB3' is included between the lox2272 site and the second continuous segment;

[0062] (iia22) contacting the recombinant genome obtained in step (iia21) with PiggyBac transposase to screen for a genome comprising the first partial segment and the loxP site located upstream of the first partial segment.

[0063] In some embodiments, in step (iib), the second partial hIgKV continuous segment of the second partial segment is inserted upstream of the first partial segment by at least the following steps, comprising:

[0064] (iib1) inserting a continuous segment comprising hIgKV2D-40 to hIgKV2D-38 genes between the mIgKJ region and the mIgKC region; and

[0065] (iib2) inserting the continuous segment between hIgKV1D-37 and hIgKV1D-35 genes and the PB3'-lox5171 and loxP sites downstream of the inserted segment in step (iib1);

[0066] (iib3) recombining the genome obtained in step (iib2) with a BAC vector containing the continuous segment between hIgKV3D-34 and hIgKV3D-7 genes and loxP and lox5171 located at both ends in the same direction as in step (iib2), and Cre recombinase, wherein PB5' is contained between the lox5171 site and the continuous segment described in this step;

[0067] (iib4) contacting the recombinant genome obtained in step (iib3) with PiggyBac transposase to screen for a genome comprising the second partial segment and the loxP site located downstream of the second partial segment.

[0068] In some embodiments, wherein step (iic) comprises:

[0069] (iic1) screening Cre-positive mice containing the first and second segments;

[0070] (iic2) mating the mice obtained in step (iic1) with wild-type mice, and screening mice that do not carry Cre but contain the first partial segment and the second partial segment, wherein there is a loxP site between the second partial segment and the first partial segment.

[0071] In some embodiments, wherein step (iid) comprises:

[0072] (iid1) mating the sixth mouse with wild-type mice to screen positive mice;

[0073] (iid2) The positive mice obtained in step (iid1) are mated between males and females to screen for homozygous mice.

[0074] Another aspect of the present invention provides a genetically modified mouse genome, wherein

[0075] The immunoglobulin heavy chain locus of the genetically modified mouse includes, in sequence: (i) the mouse Adam6a gene; (ii) the mouse Adam6b gene; (iii) the second partial segment of the human immunoglobulin heavy chain variable region locus, which includes the second partial hIgHV continuous segment; (iv) the first partial segment of the human immunoglobulin heavy chain variable region locus, which is located downstream of the second partial segment and includes the first partial hIgHV continuous segment, the entire hIgHD segment and the entire hIgHJ segment, the first partial segment does not include the segment between the downstream of the hIgHV1-2 gene and the upstream of the hIgHV6-1 gene; and (v) the mIgHC region; and

[0076] The immunoglobulin Kappa light chain genome of the genetically modified mouse includes, in sequence: (i) a second partial segment of the human immunoglobulin Kappa light chain variable region locus, which includes a second partial hIgKV continuous segment; (ii) a first partial segment of the human immunoglobulin Kappa light chain variable region locus, which is located downstream of the second partial segment and includes a first partial hIgKV continuous segment and the entire hIgHJ segment, the first partial segment and the second partial segment together constitute the entire hIgKV segment; and (iii) the mIgKC region.

[0077] In some embodiments, the first part of the hIgHV continuous segment of the first part segment can start from any hIgHV segment gene upstream of the hIgHV1-2 gene (including functional genes, pseudogenes or ORFs) and end at the hIgHV1-2 gene.

[0078] In one embodiment, the gene of any hIgHV segment upstream of the hIgHV1-2 gene can be any functional gene, pseudogene or ORF between hIgHV(III)-82 and hIgHV1-2 genes. In one embodiment, the functional gene of any hIgHV segment upstream of the hIgHV1-2 gene, such as hIgHV3-74, hIgHV3-73, hIgHV3-72, hIgHV2-70, hIgHV1-69D, hIgHV1-69-2, hIgHV2-70D, hIgHV1-69, hIgHV3-66, hIgHV3-64, hIgHV3-75, hIgHV3-76, hIgHV3-77, hIgHV3-78, hIgHV3-79, hIgHV3-80, hIgHV3-81, hIgHV3-82, hIgHV3-83, hIgHV3-84, hIgHV3-85, hIgHV3-86, hIgHV3-87, hIgHV3-88, hIgHV3-89, hIgHV3-90, hIgHV3-91, hIgHV3-92, hIgHV3-93, hIgHV3-94, hIgHV3-95, hIgHV3-96, hIgHV3-97, hIgHV3-98, hIgHV3-99, hIgHV3-91, hIgHV3-91, hIgHV3-99, hIgHV3-91, hIgHV3-91, hIgHV3-91, hIgHV3- gHV3-62, hIgHV4-61, hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgHV3- 48. hIgHV1-46, hIgHV1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3-35 , hIgHV4-34, hIgHV3-33, hIgHV4-31, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4-3 0-2, hIgHV4-30-1, hIgHV3-30, hIgHV4-28, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3-2 3. hIgHV3-21, hIgHV3-20, hIgHV1-18, hIgHV3-15, hIgHV3-13, hIgHV3-11, hIgHV5-10-1, h IgHV3-9, hIgHV3-64D, hIgHV3-8, hIgHV3-7, hIgHV2-5, hIgHV7-4-1, hIgHV4-4 or hIgHV1-3.

[0079] In one embodiment, the first portion of the first segment of the continuous segment of hlgHV starts with hlgHV3-74, hlgHV3-73, hlgHV3-72, hlgHV2-70, hlgHV1-69D, hlgHV1-69-2, hlgHV2-70D, hlgHV1-69, hlgHV3-66, hlgHV3-64, hlgHV3-62, hlgHV4- 61. hIgHV4-59, hIgHV1-58, hIgHV3-53, hIgHV5-51, hIgHV3-49, hIgHV3-48, hIgHV1-46, hIgHV 1-45, hIgHV3-43, hIgHV4-39, hIgHV3-43D, hIgHV4-38-2, hIgHV3-35, hIgHV4-34, hIgHV3-33, hIgHV4-31, hIgHV3-30-5, hIgHV4-30-4, hIgHV3-30-3, hIgHV4-30-2, hIgHV4-30-1, hIgHV3- 30. hIgHV4-28, hIgHV2-26, hIgHV1-24, hIgHV3-23D, hIgHV3-23, hIgHV3-21, hIgHV3-20, hIgH V1-18, hIgHV3-15, hIgHV3-13, hIgHV3-11, hIgHV5-10-1, hIgHV3-9, hIgHV3-64D, hIgHV3-8, hIgHV3-7, hIgHV2-5, hIgHV7-4-1, hIgHV4-4 or hIgHV1-3, terminated at the hIgHV1-2 gene, all comprising the described genes as ends. In one embodiment, the first portion of the first segment is a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0080] In the present invention, the second portion segment is located upstream of the first portion segment, and the second portion hIgHV continuous segment can start from any gene (including functional gene, pseudogene or ORF) of the hIgHV segment and terminate at a gene (including functional gene, pseudogene or ORF) of any hIgHV segment upstream of the starting gene of the first portion hIgHV continuous segment. In some embodiments, the second portion segment, together with the first portion segment, constitutes all functional genes upstream of hIgHV1-2 and the continuous segment therebetween, i.e., the continuous segment between hIgHV3-74 and hIgHV1-2 genes. For example, in some embodiments, the second portion hIgHV continuous segment includes or is a continuous segment between the hIgHV3-74 gene and the hIgHV3-30 gene, and the first portion hIgHV continuous segment includes or is a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene.

[0081] In the present invention, the first part of the hIgKV continuous segment of the first part segment of the human immunoglobulin Kappa light chain variable region locus can start from any hIgKV segment gene upstream of the hIgKV4-1 gene (including functional genes, pseudogenes or ORFs) and end at the hIgKV4-1 gene.

[0082] In one embodiment, the gene of any hIgKV segment upstream of the hIgKV4-1 gene can be any functional gene, pseudogene or ORF between the hIgKV3D-7 and hIgKV4-1 genes. In one embodiment, any gene of the hIgKV segment upstream of the hIgKV4-1 gene is a functional gene or ORF, such as hIgKV3D-7, hIgKV1D-8, hIgKV1D-43, hIgKV1D-42, hIgKV3D-11, hIgKV1D-12, hIgKV1D-13, hIgKV3D-15, hIgKV1D-16, hIgKV1D-17, hIgKV6D-41, hIgKV3D-20, hIgKV6D-21, hIgKV2D-24, hIgKV2D-26, hIgKV2D-28, hIgKV2D-29, hIgKV2D-30, hIgKV1D-33 , hIgKV1D-37, hIgKV1D-39, hIgKV2D-40, hIgKV2-40, hIgKV1-39, hIgKV1-37, hIgKV1-33, hIgKV2-30, hIgKV2-29, hIgKV2-28, hIgKV2-27, hIgKV2-24, hIgKV 6-21, hIgKV3-20, hIgKV1-17, hIgKV1-16, hIgKV3-15, hIgKV1-13, hIgKV1-12 , hIgKV3-11, hIgKV1-9, hIgKV1-8, hIgKV3-7, hIgKV1-6, hIgKV1-5 or hIgKV5-2.

[0083] In one embodiment, the first portion of the first segment of the continuous segment starts with hIgKV3D-7, hIgKV1D-8, hIgKV1D-43, hIgKV1D-42, hIgKV3D-11, hIgKV1D-12, hIgKV1D-13, hIgKV3D-15, hIgKV1D-16, hIgKV1D-17, hIgKV1D-18, hIgKV1D-20, hIgKV1D-21, hIgKV1D-22, hIgKV1D-23, hIgKV1D-24, hIgKV1D-25, hIgKV1D-26, hIgKV1D-27, hIgKV1D-28, hIgKV1D-29, hIgKV1D-30, hIgKV1D-31, hIgKV1D-32, hIgKV1D-33, hIgKV1D-34, hIgKV1D-35, hIgKV1D-36, hIgKV1D-37, hIgKV1D-38, hIgKV1D-39, hIgKV1D-40, hIgKV1D-41, hIgKV1D-42 IgKV1D-17, hIgKV6D-41, hIgKV3D-20, hIgKV6D-21, hIgKV2D-24, hIgKV2D-26, h IgKV2D-28, hIgKV2D-29, hIgKV2D-30, hIgKV1D-33, hIgKV1D-37, hIgKV1D-39, hI gKV2D-40, hIgKV2-40, hIgKV1-39, hIgKV1-37, hIgKV1-33, hIgKV2-30, hIgKV2- 29. hIgKV2-28, hIgKV2-27, hIgKV2-24, hIgKV6-21, hIgKV3-20, hIgKV1-17, hIg One of hIgKV1-16, hIgKV3-15, hIgKV1-13, hIgKV1-12, hIgKV3-11, hIgKV1-9, hIgKV1-8, hIgKV3-7, hIgKV1-6, hIgKV1-5, or hIgKV5-2, terminates at the hIgKV4-1 gene, and all contain the described genes as ends. In one embodiment, the first portion of the first segment is a continuous segment between the hIgKV1-37 gene and the hIgKV4-1 gene.

[0084] In the present invention, the second portion of the kappa light chain variable region genome is located upstream of the first portion of the kappa light chain variable region genome, and the second portion of the hIgKV continuous segment can start at any gene (including functional genes, pseudogenes, or ORFs) of the hIgKV segment and end at any gene (including functional genes, pseudogenes, or ORFs) of the hIgKV segment upstream of the starting gene of the first portion of the hIgKV continuous segment. In some embodiments, the second portion of the segment and the first portion of the segment together constitute all functional genes upstream of hIgKV4-1 and the continuous segment therebetween, i.e., the continuous segment between the hIgKV4-1 and hIgKV3D-7 genes. For example, in some embodiments, the second part of the hIgKV continuous segment includes or is a continuous segment between the hIgKV3D-7 gene and the hIgKV2D-40 gene, and the first part of the hIgKV continuous segment includes or is a continuous segment between the hIgKV1-37 gene and the hIgKV4-1 gene.

[0085] In some embodiments, the first portion segment and the second portion segment of the heavy chain and the kappa light chain are not rearranged.

[0086] In some embodiments, the immunoglobulin heavy chain locus of the obtained genetically modified mouse includes, in sequence: (i) the mouse Adam6a gene; (ii) the mouse Adam6b gene; (iii) a continuous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (iv) a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (v) a continuous segment between the hIgHV6-1 gene and the hIgHJ6 gene; (vi) the mIgHC region; and the immunoglobulin Kappa light chain locus of the obtained genetically modified mouse includes, in sequence: (i) a continuous segment between the hIgKV3D-7 gene and the hIgKV4-1 gene; (ii) a continuous segment between the hIgKJ1 gene and the hIgKJ5 gene; and (iii) the mIgKC region.

[0087] In some embodiments, the immunoglobulin heavy chain locus of the obtained genetically modified mouse does not include all mIgHV segments upstream of mIgHV5-1 and its gene, the continuous segment between the mIgHD1-1 gene and the mIgHJ4 gene, and the mIgHD3-1, mIgHD5-1 and mIgHD1-3 genes; and the immunoglobulin Kappa light chain locus of the obtained genetically modified mouse does not include the continuous segment between the mIgKV2-137 gene and the mIgKVJ5 gene.

[0088] In some embodiments, the fertility of the resulting genetically modified mice is not reduced compared to non-genetically modified wild-type mice.

[0089] In some embodiments, the obtained genetically modified mice are capable of producing human-mouse chimeric antibodies, wherein the chimeric antibodies comprise a human heavy chain variable region and a mouse heavy chain constant region, and a human kappa light chain variable region and a mouse kappa light chain constant region.

[0090] Another aspect of the present invention provides a cell, tissue, organ or mouse comprising the above-mentioned mouse genome.

[0091] In some embodiments, the present invention provides a cell comprising the above-mentioned mouse genome, wherein the cell is an embryonic cell, a B cell, or a hybridoma cell.

[0092] In some embodiments, the present invention provides a tissue comprising the above-mentioned mouse genome, wherein the tissue is the white pulp of the spleen or its lymphoid nodules.

[0093] In some embodiments, the present invention provides an organ comprising the above-mentioned mouse genome, wherein the organ is a spleen.

[0094] In some embodiments, the present invention provides a mouse comprising the above-described mouse genome.

[0095] Another aspect of the present invention provides a method for preparing a monoclonal antibody, the method comprising

[0096] (a) immunizing a mouse having any one of the genomes described in the present invention with an antigen;

[0097] (b) isolating cells producing monoclonal antibodies against the antigen from the mouse; and

[0098] (c) culturing the cells to obtain the monoclonal antibody.

[0099] In some embodiments, the cell of step (c) is a spleen cell, a B cell, or a hybridoma cell.

[0100] In some embodiments, the monoclonal antibody has a human heavy chain variable region and a human kappa light chain variable region, and does not have a mouse heavy chain variable region and a mouse kappa light chain variable region. In some embodiments, the monoclonal antibody has a human heavy chain variable region, a human kappa light chain variable region, a mouse heavy chain constant region, and a mouse kappa light chain constant region.

[0101] Another aspect of the present invention provides use of any cell, tissue, organ or mouse of the present invention in preparing monoclonal antibodies.

[0102] In some embodiments, the monoclonal antibody has a human heavy chain variable region and a human kappa light chain variable region, and does not have a mouse heavy chain variable region and a mouse kappa light chain variable region. In some embodiments, the monoclonal antibody has a human heavy chain variable region, a human kappa light chain variable region, a mouse heavy chain constant region, and a mouse kappa light chain constant region.

[0103] The present invention provides a mouse genome with a modified heavy chain locus that retains the endogenous ADAM6 gene and its function, enabling the production of human-mouse chimeric antibodies while maintaining fertility. The chimeric antibodies comprise a human heavy chain variable region, a human kappa light chain variable region, a mouse heavy chain constant region, and a mouse kappa light chain constant region. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figures 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 34, 35, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, and 82 respectively represent schematic diagrams of the genomic changes involved in each modification step.

[0105] Figures 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 37, 38, 39, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 84, 85 and 86 respectively represent the results of PCR identification of each modification step.

[0106] 40 to 42 show the usage frequency distribution diagrams of genes at the reads level based on the obtained gene usage frequency data.

[0107] FIG43 shows a distribution diagram of CDR3 amino acid length at the reads level based on the obtained CDR3 length frequency data.

[0108] FIG44 shows the Weblogo feature analysis results of the CDR3 amino acid sequences of each sample at the reads level.

[0109] Figure 45 shows that genetically modified mice and wild-type mice have similar immune properties.

[0110] FIG46 shows a representative detection case of positive hybridoma cells obtained by screening.

[0111] FIG47 shows that dozens of positive hybridoma cells were screened, of which 5 had very strong binding ability to antigen-positive cells.

[0112] Figure 48 shows that the four gene-modified homozygous mice all exhibited higher immune response capabilities.

[0113] Figure 49 shows that the antibody sequences obtained from genetically modified mice are all human antibody sequences and have rich sequence diversity.

[0114] Figures 87 and 88 show the usage frequency distribution diagrams of IgGKV region genes and IgGKJ region genes at the reads level based on the obtained gene usage frequency data.

[0115] FIG89 shows a distribution diagram of CDR3 amino acid length at the reads level based on the obtained CDR3 length frequency data.

[0116] Figures 90 to 93 are PCR identification diagrams of heavy chain and kappa light chain double gene heterozygous mice.

[0117] Figures 94 to 97 are PCR identification diagrams of mice homozygous for both heavy chain and kappa light chain genes. DETAILED DESCRIPTION

[0118] definition

[0119] "hIgHV" as used herein refers to the V region of the human immunoglobulin heavy chain variable region locus. When used independently, it refers to the entire V region of the human immunoglobulin heavy chain variable region locus. When it is suffixed with a specific gene number, for example, "hIgHV3-30" refers to the 3-30 genes located in the V region of the human immunoglobulin heavy chain variable region locus. Similarly, "hIgHD" and "hIgHJ" are also used herein to refer to the D region and J region of the human immunoglobulin heavy chain variable region locus, respectively.

[0120] "hIgKV" as used herein refers to the V region of the human immunoglobulin kappa light chain variable region locus. When used independently, it refers to the entire V region of the human immunoglobulin kappa light chain variable region locus. When it is suffixed with a specific gene number, for example, "hIgKV1-37" refers to genes 1-37 located in the V region of the human immunoglobulin kappa light chain variable region locus. Similarly, "hIgKJ" is also used herein to refer to the J region of the human immunoglobulin kappa light chain variable region locus.

[0121] "mIgHV" in the present invention refers to the V region of the mouse immunoglobulin heavy chain variable region locus. When used independently, it refers to the entire V region of the mouse immunoglobulin heavy chain variable region locus. When it is suffixed with a specific gene number, for example, "mIgHV5-1" refers to the 5-1 gene located in the V region of the mouse immunoglobulin heavy chain variable region locus. Similarly, the present invention also uses "mIgHD" and "mIgHJ" to refer to the D region and J region of the mouse immunoglobulin heavy chain variable region locus, respectively.

[0122] "mIgKV" in the present invention refers to the V region of the mouse immunoglobulin kappa light chain variable region locus. When used independently, it refers to the entire V region of the mouse immunoglobulin kappa light chain variable region locus. When it is suffixed with a specific gene number, for example, "mIgKV2-137" refers to the 2-137 gene located in the V region of the mouse immunoglobulin kappa light chain variable region locus. Similarly, "mIgKJ" is also used in the present invention to refer to the J region of the mouse immunoglobulin kappa light chain variable region locus.

[0123] "Continuous segment" refers to a non-interrupted nucleotide segment between two designated endpoint genes, which includes functional genes, pseudogenes, ORFs, and other nucleotide sequences (such as spacer sequences) located between the two endpoint genes. The term "continuous segment between gene A and gene B" refers to a continuous gene segment including gene A, gene B, and the segment between the two. The terms "upstream of gene A" or "downstream of gene A" do not include gene A itself.

[0124] Example

[0125] Example 1. Preparation of heavy chain gene locus modified mice

[0126] 1. Construction of mice based on ES cell line I

[0127] 1. Contains approximately 20kb of human gene sequence (including IGHJ1-6, IGHD7-27, IGHD1-26, IGHD6-25 and all intergenic sequences) inserted between the mouse J region and C region

[0128] The constructed vector was electroporated into wild-type ES cells. The vector carries Neo resistance. The cells were screened with G418 drug, and relevant ES clones were selected for culture and amplification, followed by PCR typing and identification.

[0129] The sequences were inserted into the corresponding positions using four pairs of primers (sequences are shown in Table 1 below, 5'→3', the same below; the primer identification positions are also marked on the map (Figure 1)). Three clones amplified positive bands simultaneously with the four pairs of primers (Figure 2): 1A3, 1A7, and 1B7. These three clones were used as positive clones in the first step.

[0130] Table 1.

[0131] 2. Insert approximately 20kb of human D genome sequence (including IGHD5-24, IGHD4-23, IGHD3-22, IGHD2-21, IGHD1-20, IGHD6-19, IGHD5-18, IGHD4-17, IGHD3-16, IGHD2-15, IGHD1-14, IGHD6-13 and all intergenic sequences) before the cell genome obtained in the first step.

[0132] The constructed vector was electroporated into the positive 1A7 clone from the first step. The vector carries Puro resistance, and one homology arm is attached to the human sequence from the positive clone from the first step, thus eliminating Neo resistance during homologous recombination. The cells were then screened with Puromycin, and relevant ES clones were selected for culture, expansion, and PCR typing.

[0133] Four primer pairs (sequences shown in Table 2 below, with primer identification positions also noted on the map (Figure 3)) were used to identify the sequence insertions at the corresponding positions. Three clones (Figure 4) amplified positive bands using all four primer pairs: 1A7-1C2, 1A7-1C3, and 1A7-1B4. These three clones were selected as positive clones in the second step.

[0134] Table 2.

[0135] 3. Insert approximately 20kb of human D genome sequence (including IGHD5-12, IGHD4-11, IGHD3-10, IGHD3-9, IGHD2-8, IGHD1-7, IGHD6-6, IGHD5-5, IGHD4-4, IGHD3-3, IGHD2-2, IGHD1-1 and all intergenic sequences) before the cell genome obtained in the second step.

[0136] The constructed vector was electroporated into the positive cell 1A7-1C3 clone from the second step: the vector carries Neo resistance, and one end of the homology arm is set on the human sequence of the positive clone from the second step, so Puro resistance will be lost due to homologous recombination in this step; the cells were screened with G418 drug, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0137] Four primer pairs (sequences shown in Table 3 below, with primer identification positions also noted on the map (Figure 5)) were used to identify the sequence insertions at the corresponding positions. Four clones (Figure 6) amplified positive bands using all four primer pairs: 1A7-1C3-1B1, 1A7-1C3-1C6, 1A7-1C3-1C9, and 1A7-1C3-1F10. These four clones were designated as positive clones in the third step.

[0138] Table 3.

[0139] 4. Insert about 20kb of human V genome sequence (including all the spacer sequences between IGHV6-1 and IGHD1-1 upstream) in front of the cell genome obtained in step 3, and introduce two lox sites loxp-lox2272 at the same time

[0140] The constructed vector was electroporated into the positive cells 1A7-1C3-1B1 clone from the third step: the vector carries Puro resistance, and one end of the homology arm is set on the human sequence of the positive clone from the second step, so Puro resistance will be lost due to homologous recombination in this step, and the Puro resistance has loxP and lox2272-PB5' (5'ITR, 5'inverted terminal repeat) elements at both ends, respectively; the cells were screened with Puromycin drugs, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0141] Four primer pairs (sequences shown in Table 4 below, with primer identification positions also noted on the map (Figure 7)) were used to identify the sequence insertions at the corresponding positions. Three clones (Figure 8) amplified positive bands using all four primer pairs: 1A7-1C3-1B1-2C1, 1A7-1C3-1B1-2C2, and 1A7-1C3-1B1-2D5. These three clones were selected as positive clones in the fourth step.

[0142] Table 4

[0143] 5. Insert approximately 400 kb of human V genome sequence (including IGHV1-2, IGHV1-3, IGHV4-4, IGHV7-4-1, IGHV2-5, IGHV3-7, IGHV3-64D, IGHV5-10-1, IGHV3-11, IGHV3-13, IGHV3-15, IGHV3-16, IGHV1-18, IGHV3-20, IGHV3-21, IGHV3-23, IGHV1-24, IGHV2-26, IGHV4-28 and all intergenic sequences) before the cell genome obtained in step 4.

[0144] The constructed fusion BAC and Cre were electroporated into the positive cell clones from step 4, 1A7-1C3-1B1-2D5. The BACs also carried Neo resistance (Neo antibodies with inverted terminal repeats at both ends), and the human sequence on the BACs contained loxP and lox2272 elements, oriented in the same direction as the positive clones from step 4. A PB3' (3' ITR, 3' inverted terminal repeat) was located between the lox2272 element and the human genomic sequence. Cre recombinase replaced the Puro resistance between the loxP and lox2272 elements in the BACs and the positive cell clones from step 4, replacing the Puro resistance with the human genomic sequence and Neo resistance on the BACs. The cells were then selected with G418, and relevant ES clones were cultured, expanded, and subsequently identified by PCR typing.

[0145] Sequence substitutions were identified using 11 primer pairs (sequences shown in Table 5 below, with primer identification positions also noted on the map (Figure 9)). Two clones (Figure 10) simultaneously amplified positive bands using all 11 primer pairs: 1A7-1C3-1B1-2D5-2A2 and 1A7-1C3-1B1-2D5-2B3. These two clones were designated as positive clones in step 5.

[0146] Table 5

[0147] 6. Electroporate pBase (PiggyBac transposase) into the cell clones obtained in step 5

[0148] pBase (PiggyBac transposase) is electroporated into the positive ES cell clones 1A7-1C3-1B1-2D5-2B3 from step 5. PiggyBac transposase excises the two inverted terminal repeats from the genome, eliminating the Neo sequence and lox2272. The positive cell clones will contain only the inserted human gene sequence and a loxP element. Select ES clones for culture, expansion, and PCR typing.

[0149] Two pairs of primers (sequences shown in Table 6 below, with primer identification positions also noted on the map ( Figure 11 )) were used to identify the sequence deletion. Four clones ( Figure 12 ) amplified positive bands using both primer pairs: 1A7-1C3-1B1-2D5-2B3-1C2, 1A7-1C3-1B1-2D5-2B3-1D1, 1A7-1C3-1B1-2D5-2B3-1D5, and 1A7-1C3-1B1-2D5-2B3-1D6. These four clones were designated as positive clones in step 6.

[0150] Table 6.

[0151] 7. F0 mice obtained from ES1

[0152] The positive cell clones obtained in step 6 are injected into blastocysts, which are then transplanted into surrogate mother mice. After a gestation period of about 20 days, mice are born. The paws of 5-7 day old mice are cut, DNA is extracted, and PCR typing is performed to confirm the mouse genotype.

[0153] A total of 8 mice were born after the injection of the 1A7-1C3-1B1-2D5-2B3-1C2 clone. All 8 mice were positive after identification using 5 pairs of primers (Table 7) ( Figures 13 and 14 ).

[0154] Table 7.

[0155] 8.ES1-derived F1 mice

[0156] F0 mice that tested positive were mated with CMV-Cre mice to generate F1 mice. The genomic DNA from the tail of the F1 mice was subjected to PCR typing to confirm the genotype of the mice.

[0157] A total of 6 F1 mice were born. ES1 was identified by 5 pairs of primers (Table 8), and CMV-Cre was identified by 1 pair of primers. A total of 2 mice (1# and 5#) were double-gene positive mice (Figures 15 and 16).

[0158] Table 8.

[0159] 2. Construction of mice based on ES cell line II

[0160] 1. Contains approximately 20kb of human gene sequence (including IGHV3-74, IGHV3-73, IGHV3-72 and all intergenic sequences) inserted between the mouse J region and C region

[0161] The constructed vector was electroporated into wild-type ES cells. The vector carries Neo resistance. The cells were screened with G418 drug, and relevant ES clones were selected for culture and amplification, followed by PCR typing and identification.

[0162] Four primer pairs (sequences shown in Table 9 below, with primer identification positions also noted on the map (Figure 17)) were used to identify the sequence insertions at the corresponding positions. Two clones (Figure 18) amplified positive bands using all four primer pairs: 1B2 and 1H5. These two clones were considered positive clones in the first step.

[0163] Table 9.

[0164] 2. Insert approximately 20 kb of human V genome sequence (including IGHV2-70, IGHV1-69D and all intergenic sequences) downstream of the cell genome obtained in the first step, and introduce two lox sites loxp-lox5171 at the same time.

[0165] The constructed vector was electroporated into the positive cell 1H5 clone from the first step: the vector carries Puro resistance, and one end of the homology arm is set on the human sequence of the positive clone from the first step, so Neo resistance will be lost due to homologous recombination in this step, and the Puro resistance ends carry PB3' (3'ITR, 3'inverted terminal repeat, 3' reverse terminal repeat)-lox5171 and loxP elements respectively; the cells were screened with Puromycin drug, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0166] Four primer pairs (sequences shown in Table 10 below, with primer identification positions also noted on the map (Figure 19)) were used to identify the sequence insertions at the corresponding positions. Two clones (Figure 20) amplified positive bands using all four primer pairs: 1H5-1B1 and 1H5-1E3. These two clones were selected as positive clones in the second step.

[0167] Table 10.

[0168] 3. Insert 400kb of human V genome sequence (including IGHV1-69-2, IGHV2-70D, IGHV1-69, IGHV3-66, IGHV3-64, IGHV4-61, IGHV4-59, IGHV1-58, IGHV3-53, IGHV8-5-51, IGHV5-51, IGHV3-49, IGHV3-48, IGHV1-46, IGHV1-45, IGHV3-43, IGHV4-39, IGHV3-38, IGHV3-35, IGHV4-34, IGHV3-33, IGHV4-31, IGHV3-30 and all intergenic sequences) downstream of the cell genome obtained in the second step.

[0169] The constructed fusion BAC and Cre were electroporated into the positive cell 1H5-1B1 clone in the second step: the BAC also carried Neo resistance (Neo resistance had inverted terminal repeat sequences at both ends), and the human sequence on the BAC had loxP and lox5171 elements at both ends in the same direction as the positive clone in the second step, and there was PB5' (5'ITR, 5'inverted terminal repeat, 5' inverted terminal repeat) between the lox5171 element and the human genomic sequence. Under the action of Cre recombinase, the sequences between loxP and lox5171 in the BAC and the positive cell clone in the second step were replaced, and Puro resistance was replaced by the human genomic sequence and Neo resistance on the BAC; the cells were screened with G418 drugs, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0170] Sequence substitutions were identified using 11 primer pairs (sequences shown in Table 11 below, with primer identification positions indicated on the map (Figure 21)). Three clones (Figure 22) simultaneously amplified positive bands using all 11 primer pairs: 1H5-1B1-1A6, 1H5-1B1-1B4, and 1H5-1B1-1E2. These three clones were selected as positive clones in the third step.

[0171] Table 11.

[0172] 4. Electrotransfect pBase (PiggyBac transposase) into the cell clones obtained in step 3

[0173] pBase (PiggyBac transposase) is electroporated into the positive cell clone 1H5-1B1-1A6 from step 3. Under the action of PiggyBac transposase, the two inverted terminal repeat sequences will be cut out from the genome, the Neo sequence and lox5171 will be lost, and the positive cell clone will only carry the inserted human gene sequence and a loxP element. ES clones are selected for culture, amplification, and PCR typing.

[0174] Three primer pairs (sequences shown in Table 12 below, with primer identification positions also noted on the map ( Figure 23 )) were used to identify the sequence deletion. Five clones ( Figure 24 ) amplified positive bands using all three primer pairs: 1H5-1B1-1A6-1B7, 1H5-1B1-1A6-1C8, 1H5-1B1-1A6-1D4, 1H5-1B1-1A6-1E8, and 1H5-1B1-1A6-1F5. These five clones were designated as positive clones in the third step.

[0175] Table 12.

[0176] 5. ES2 F0 mice

[0177] The positive cell clones obtained in the fourth step are injected into blastocysts, which are then transplanted into surrogate mother mice. After a gestation period of about 20 days, mice are born. The paws of 5-7 day old mice are cut, DNA is extracted, and PCR typing is performed to confirm the mouse genotype.

[0178] A total of 8 mice were born after the injection of the 1H5-1B1-1A6-1C8 clone. All 8 mice were positive after joint identification using 4 pairs of primers (Table 13) ( Figures 25 and 26 ).

[0179] Table 13.

[0180] 6.ES2-derived F1 mice

[0181] The F0 mice that tested positive were mated with wild-type mice to obtain F1 mice. The genomic DNA of the F1 mice's tails was subjected to PCR typing to confirm the genotype of the mice.

[0182] A total of 6 F1 mice were born, and ES2 was jointly identified by 4 pairs of primers (Table 14). A total of 3 mice (2#, 5#, and 6#) were double-gene positive mice (Figures 27 and 28).

[0183] Table 14.

[0184] ES1 double-gene positive F1 mice were hybridized with ES2-derived F1-positive mice to obtain Cre-positive, ES1-positive and ES2-positive F2 mice; the obtained F2-positive mice were mated with wild-type mice to obtain 3.5-day blastocysts, and ES cell lines were established. Different ES cell lines were tested by PCR to screen out cells that did not carry Cre but had undergone ectopic recurrence. These cells covered the human genome sequences of ES1 and ES2, and had a loxP sequence between IGHV3-30 and IGHV4-28.

[0185] Three pairs of primers (Table 15) were used to identify translocation of the gene. Four clones were identified as translocated (Figures 29 and 30): 1A3, 1A5, 1A6, and 1B3. These four clones were designated as ES cell line III.

[0186] Table 15.

[0187] 3. Gene Knockout (KO)

[0188] 1. Perform the first homologous recombination KO (V region) on ES cell line III

[0189] A vector was constructed. The vector was conferring Neo resistance (with recombinase sites at both ends of Neo). The 5-terminal homology arm sequence contained a 10kb continuous sequence upstream of the mIgHV1-86 gene, and the 3-terminal homology arm sequence contained an 8kb continuous sequence downstream of the mIgHV5-2 gene. The constructed vector was electroporated into ES cell line III. The cells were screened with G418 drug, and relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0190] Two primer pairs (sequences shown in Table 16 below, with primer identification positions indicated on the map (Figure 31)) confirmed that the V region genes (mIgHV1-86 to mIgHV5-2) had undergone Neo-resistance recombination, indicating a recombinant deletion in the mouse IgH chain V gene. Eight clones (Figure 32) amplified positive bands using both primer pairs: 1A3-1B3, 1A3-1B4, 1A3-1B5, 1A3-1B7, 1A3-1E1, 1A3-1E2, 1A3-1E3, and 1A3-1E5. These eight clones were considered positive clones in the first step.

[0191] Table 16. First step KO ES cells

[0192] 2. The cells obtained in the first step were subjected to a second homologous recombination KO (knockout of all gene sequences between IgHJ4 and IgHD1-1)

[0193] A vector was constructed. The vector was conferred with Puro resistance (with recombinase sites at both ends of Puro). The 5-terminal homology arm sequence contained an 8kb continuous sequence upstream of the mIgHD1-1 gene, and the 3-terminal homology arm sequence contained a 5kb continuous sequence downstream of the mIgHJ4 gene. The constructed vector was electroporated into the positive cell clones 1A3-1B3 from the first step. The cells were screened with Puromycin, and the relevant ES clones were selected for culture, amplification, and PCR typing.

[0194] Two primer pairs (sequences shown in Table 17 below, with primer identification positions indicated on the map (Figure 33)) confirmed Puro-resistant recombination between the mIgHJ4 and mIgHD1-1 genes, indicating a recombinant deletion between the mIgHJ4 and mIgHD1-1 genes in the mouse IgH chain. Three clones amplified positive bands using both primer pairs (Figure 34): 1A3-1B3-1C3, 1A3-1B3-1C6, and 1A3-1B3-1C8. These three clones were selected as positive clones in the second step.

[0195] Table 17. 1A3-1B3 Second Step KO ES Cells

[0196] 3. The cells obtained in the second step were subjected to the third homologous recombination KO (knockout of mIgHD3-1, mIgHD5-1 and mIgHD1-3 genes)

[0197] A vector was constructed that confers Hygro resistance (with recombinase sites at both ends of the Hygro vector). The 5'-terminal homology arm sequence contained an 8kb continuous sequence upstream of the mIgHD1-3 gene, and the 3'-terminal homology arm sequence contained a 5kb continuous sequence downstream of the mIgHD3-1 gene. The constructed vector was electroporated into the positive clones 1A3-1B3-1C6 from the second step. The cells were screened with Hygromycin B, and the relevant ES clones were selected for culture, amplification, and PCR typing.

[0198] Two primer pairs (sequences shown in Table 18 below, with primer identification positions indicated on the map (Figure 35)) confirmed that the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes had undergone Hygro-resistance recombination, indicating that the mouse IgH chain mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes had undergone recombination deletion. Three clones (Figure 36) amplified positive bands using both primer pairs: 1A3-1B3-1C6-1D2, 1A3-1B3-1C6-1D5, and 1A3-1B3-1C6-1D8. These three clones were designated as positive clones in the third step.

[0199] Table 18. 1A3-1B3-1C6 Step 3 KO ES Cells

[0200] 4. Inject the positive cell clones obtained in the third step to obtain positive F0 mice

[0201] The positive cell clones obtained in the third step are injected into blastocysts, which are then transplanted into surrogate mother mice. After a gestation period of about 20 days, mice are born. The paws of 5-7 day old mice are cut, DNA is extracted, and PCR typing is performed to confirm the mouse genotype.

[0202] A total of 18 mice were born after the injection of the 1A3-1B3-1C6-1D8 clone. After joint identification by three pairs of primers (Table 19), three mice were positive: 3#15#18# (Figure 37).

[0203] Table 19. ES cells after KO - 1A3-1B3-1C6-1D8 injected into F0 mice

[0204] 5. Obtaining Positive F1 Mice

[0205] F0 mice that tested positive were mated with wild-type mice to obtain F1 mice. The genomic DNA of the F1 mice's tails was subjected to PCR typing to confirm the genotype of the mice.

[0206] A total of 16 F1 mice were born, and three mice (2#6#16#) were identified as double-gene positive mice by co-identification using three pairs of primers (Table 20) (Figure 38).

[0207] Table 20. F0-3# mice bred with WT to obtain F1 mice

[0208] 6. Obtaining Homozygous Mice

[0209] Homozygous mice were obtained by mating the F1 mice that were positive. The genomic DNA of the F2 generation mice was used for PCR typing to confirm the genotype of the mice.

[0210] A total of 12 F2 mice were born. After joint identification with 6 pairs of primers (Table 21), it was determined that the genotypes of mice #1, 3, and 4 were homozygous; the genotypes of mice #2, 5, 6, 11, and 12 were heterozygous; and the genotypes of mice #7, 8, 9, and 10 were wild type (Figure 39).

[0211] Table 21. F1-2#♂ crossbreeding with F1-16#♀

[0212] 4. Breeding performance testing

[0213] The homozygous mice obtained were further bred and their reproductive performance was analyzed.

[0214] A total of 25 breeding pairs were bred, and the number of mice born one month after breeding was counted. Of the 25 breeding pairs, 20 gave birth one month after breeding, representing an effective reproductive number of 80%. The average litter size was 5.35 mice, consistent with the litter size of wild-type mice. The data are shown in Table 22.

[0215] Table 22.

[0216] 5. H chain immunohistochemistry sequencing

[0217] The homozygous mice were euthanized and their spleens were dissected for RNA extraction. After the total RNA of the samples passed the test, library construction was performed, followed by immune repertoire sequencing (immune repertoire sequencing (Immuno-Seq) is a study of B / T lymphocytes. It uses 5'RACE or multiplex PCR technology to specifically amplify the variable regions (V regions) that determine the diversity of B cell receptors (BCR) or T cell receptors (TCR), combined with high-throughput sequencing technology to comprehensively assess the diversity of the immune system). The sequenced sequences were quality controlled using quality control software and the sequencing background was filtered. They were then compared with the V, D, and J genes of the IMGT immune cell receptor library to search for corresponding gene fragments, find the precise sites of V, D, and J gene fragments and sequences, and statistically analyze information such as V, D, and J gene frequencies, clone frequency distribution, and number of polypeptide sequences.

[0218] Figures 40 to 42 show the frequency distribution of gene usage at the read level based on the obtained gene usage frequency data. The results in Figure 40 show that the usage frequency of IGHV43-34 in unimmunized homozygous mice is the highest at the read level, followed by IGHV6-1, IGHV4-59, IGHV4-39, IGHV3-21, IGHV2-26, IGHV3-23, IGHV2-5, IGHV5-51, IGHV1-18, IGHV3-48, IGHV3-15, IGHV4-4, IGHV3-43, IGHV5-10-1, IGHV3-30, IGHV3-7, IGHV1-3, IGHV4-61, IGHV3-20, IGHV3-74, IGHV1-69, IGHV3-73, IGHV1-2, IGHV7-4-1, IGHV7-5-2 I GHV3-43D, IGHV3-38, IGHV3-33, IGHV2-70, IGHV3-71, IGHV3-NL1, IGHV3-22, IGHV3-30-3, IGHV3-38-3, IGHV3-35, IGHV3-52, IGHV4-31, IGHV4 / OR15-8 ​​in descending order. The results in Figure 41 show that IGHD3-10 of unimmunized homozygous mice has the highest usage frequency at the read level, followed by IGHD6-13, IGHD6-19, IGHD3-9, IGHD1-26, IGHD7-27, IGHD5-12, IGHD4-17, IGHD5-18, IGHD2-2, IGHD1-1, IGHD2-15, IGHD3-22, IGHD3-16, IGHD2-21, IGHD4-23, IG The frequency of use of IGHJ4 in unimmunized homozygous mice at the read level was highest, followed by IGHJ6, and then IGHJ3, IGHJ5, IGHJ2, and IGHJ1, respectively.

[0219] Figure 43 shows the distribution of CDR3 amino acid length at the read level based on the obtained CDR3 length frequency data. The results show that the distribution of CDR3 amino acid length is close to a normal distribution.

[0220] CDR3 amino acid sequence feature analysis is a crucial step in immunohistochemistry analysis. By refining the analysis down to each clone, we quantitatively analyze the abundance of the different CDR3 sequences that evolved from each germline antibody sequence, ultimately facilitating the selection of sequences for further antibody expression. The Weblogo feature analysis of the CDR3 amino acid sequences of each sample at the read level is shown in Figure 44. The results in Figure 44 show the most abundant amino acid sequences in the CDR3 of unimmunized homozygous mice.

[0221] VI. Antibody Preparation

[0222] Three 6-8 week old C57BL / 6N female mice and three 6-8 week old HK homozygous female mice (mice homozygous for humanized heavy chain variable regions according to steps 1 to 3 above, and humanized kappa light chain variable regions) were immunized with B7-H3-CHO-K1 stable cell lines. The immunization regimen consisted of three routine immunizations plus one booster immunization. The three routine immunizations were performed every two weeks, with mice injected intraperitoneally with 2×10 7 B7-H3-CHO-K1 cells. Three days after the third routine immunization, peripheral blood was collected from the mice through orbital bleeding, and serum was collected. The antibody titer of the mouse serum was detected by flow cytometry. After the titer was qualified, a booster immunization was performed. The booster immunization was performed 10 days after the third routine immunization, and the immunization method was intraperitoneal injection of 2×10 7 B7-H3-CHO-K1 cells. Three days after the booster immunization, the mouse spleens were harvested for hybridoma fusion to generate hybridoma cells. After hybridoma cell growth, positive hybridoma cells were identified: supernatants were collected and analyzed by flow cytometry for the presence of B7-H3 antibodies. The experimental results showed that the immune performance of HK homozygous mice was comparable to that of wild-type C57BL / 6N mice. After three immunizations, serum antibody titers of the two were comparable, with the highest titer exceeding 1:256,000. Subsequently, spleen cells from HK mice were fused with myeloma SP20 cells to generate hybridoma cells. Through multiple rounds of screening, over 70 B7-H3 antibody-positive hybridoma cells were obtained, of which five hybridoma cells secreted antibodies that had very strong binding ability to antigen-positive cells.

[0223] After booster immunization, mouse serum was collected for antibody titer testing. The serum dilution gradient was set as 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. The test results in Figure 45 show that HK mice and wild-type mice have similar immune properties, and the serum titers of both are >1:256000.

[0224] After hybridoma cell fusion, the culture supernatant of the successfully fused cells is selected and co-incubated with antigen-positive cells to identify positive hybridoma cells that can secrete antibodies. Figure 46 shows a representative detection example of positive hybridoma cells obtained through screening.

[0225] The average fluorescence intensity of the binding of positive hybridoma cell supernatants to antigen-positive cells was statistically analyzed. The results in Figure 47 show that dozens of positive hybridoma cells were screened, of which 5 had very strong binding ability to antigen-positive cells: 5H9, 3A10, 6F12, 325-1G10, and 7D5.

[0226] To generate a humoral immune response against PD-L1 in HK homozygotes (mice homozygous for humanized heavy and kappa light chain variable regions), the first immunization consisted of a mixture of 0.05 mg of PD-L1 His protein (Kaika Biotechnology, PDL-HM110) in Freund's complete adjuvant (CFA) and subcutaneous injection. Subsequently, 0.025 mg of PD-L1 His protein in incomplete Freund's adjuvant (IFA) was subcutaneously injected. Immunizations were repeated four times, every two weeks, to allow HK homozygous mice to produce antigen-specific antibodies. Serum titers were measured using serum from HK homozygous mice after the third and fourth immunizations. The cells were coated with 2 μg / ml of antigen overnight in carbonate-buffered saline (CBS), washed with phosphate-buffered saline (PBST), and then blocked with 2% BSA at 37°C for 2 hours. After washing with PBST, serum dilutions (eight serial dilutions, starting at 1:2000) were added and blocked at 37°C for 2 hours. After washing with PBST, horseradish peroxidase-labeled goat anti-mouse secondary antibody (diluted 1:1 with PBS) was added and incubated at 37°C for 1 hour. After washing with PBST, TMB colorimetric solution (Biyuntian, P0209-500ml) was added for color development at 37°C for 5-10 minutes. The reaction was terminated by adding stop solution, and the serum titer was measured by optical density at 450 nm. The results are shown in Figure 48. All four HK homozygous mice showed a high immune response capacity, reaching above 256K (HK-84, HK-90, HK-106, and HK-110 are HK homozygous mice; WT-C57B6 is a wild-type mouse; NC is the serum of non-immunized mice as a negative control).

[0227] The spleen of the mouse with the highest serum titer after four rounds of immunization was collected, and the cells were collected after grinding. Total RNA from spleen cells was extracted using an RNA extraction kit (Fuji Bio, RE-03011). cDNA was synthesized and the antibody VH and VL sequences were amplified using slot PCR. The vector pComb3XSS (Edi Gene, 63890) and the target fragment were digested and recovered with SfiI, and the ligation product was electroporated into TG1 competent cells to construct a PD-L1 scFv antibody library and measure the library capacity. The library capacity was 1.36×109 CFM. 48 clones were randomly selected from the library transformant number titer plate for sequencing identification. The results (Figure 49) showed that the antibody sequences were all human antibody sequences and the sequence diversity was rich.

[0228] Example 2. Preparation of Kappa Light Chain Locus Modified Mice

[0229] 1. ES cell line I

[0230] 1. Approximately 20 kb of human gene sequence (including IGKJ1-5 and all intergenic regions) is inserted between the mouse J and C regions.

[0231] The constructed vector was electroporated into wild-type ES cells. The vector carries Neo resistance. The cells were screened with G418 drug, and relevant ES clones were selected for culture and amplification, followed by PCR typing and identification.

[0232] The cell clones were amplified using three pairs of primers (sequences are shown in Table 23 below, and the primer identification positions are also marked on the map (Figure 50)), and the identification sequences were inserted into the corresponding positions.

[0233] Three clones simultaneously amplified positive bands using the three primer pairs ( FIG. 51 ): 1A4, 1A5, and 1B2. These three clones were considered positive clones in the first step.

[0234] Table 23

[0235] 2. Insert approximately 20kb of human V region genomic sequence (including IGKV4-1, IGKV5-2 and all gene intergenic sequences) before the cell genome obtained in the first step.

[0236] The constructed vector was electroporated into the positive cell 1A5 clone from the first step: the vector carries Puro resistance, and one end of the homology arm is set on the human sequence of the positive clone from the first step, so Neo resistance will be lost due to homologous recombination in this step; the cells are screened with Puromycin, and the relevant ES clones are selected for culture and amplification, and then PCR typing and identification.

[0237] The sequences were inserted into the corresponding positions using three pairs of primers (sequences are shown in Table 24 below, and the primer identification positions are also marked on the map (Figure 52)).

[0238] There were four clones that simultaneously amplified positive bands using the three pairs of primers ( FIG. 53 ): 1A5-1D5, 1A5-2A3, 1A5-2A5, and 1A5-2A8. These four clones were used as positive clones in the second step.

[0239] Table 24

[0240] 3. Insert approximately 20kb of human V region genomic sequence (including IGKV7-3 and adjacent sequences) before the cell genome obtained in the second step.

[0241] The constructed vector was electroporated into the positive cells 1A5-2A3 clones from the second step: the vector carries Neo resistance, and one end of the homology arm is set on the human sequence of the positive clone from the second step, so Puro resistance will be lost due to homologous recombination in this step; the cells were screened with G418 drug, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0242] The sequences were inserted into the corresponding positions using three pairs of primers (sequences are shown in Table 25 below, and the primer identification positions are also marked on the map (Figure 54)).

[0243] Three clones simultaneously amplified positive bands using the three primer pairs ( FIG. 55 ): 1A5-2A3-1F4, 1A5-2A3-1F12, and 1A5-2A3-1G4. These three clones were used as positive clones in the third step.

[0244] Table 25

[0245] 4. Insert approximately 20 kb of human V region genomic sequence (including IGKV2-4, IGKV1-5 and all gene intergenic sequences) in front of the cell genome obtained in the third step, and introduce two lox sites loxp-lox2272 at the same time.

[0246] The constructed vector was electroporated into the positive cells 1A5-2A3-1G4 clone from the third step: the vector carries Puro resistance, and one end of the homology arm is set on the human sequence of the positive clone from the second step, so Neo resistance will be lost due to homologous recombination in this step, and the Puro antibody has loxP and lox2272-PB5' (5'ITR, 5'inverted terminal repeat) elements at both ends, respectively; the cells were screened with Puromycin drugs, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0247] The sequences were inserted into the corresponding positions using three pairs of primers (sequences are shown in Table 26 below, and the primer identification positions are also marked on the map (Figure 56)).

[0248] Three clones simultaneously amplified positive bands using the three primer pairs ( FIG. 57 ): 1A5-2A3-1G4-1C1, 1A5-2A3-1G4-1C7, and 1A5-2A3-1G4-1E6. These three clones were used as positive clones in the fourth step.

[0249] Table 26

[0250] 5. Insert about 350 kb of human V genome sequence (including IGKV1-6, IGKV3-7, IGKV1-8, IGKV1-9, IGKV2-10, IGKV3-11, IGKV1-12, IGKV1-13, IGKV2-14, IGKV3-15, IGKV1-16, IGKV1-17, IGKV2-18, IGKV2-19, IGKV3-20, IGKV1-10, IGKV1-11, IGKV1-12, IGKV1-13, IGKV2-14, IGKV3-15, IGKV1-16, IGKV1-17, IGKV2-18, IGKV2-19, IGKV2-10, IGKV3-20, IGKV1-10, IGKV1-11, IGKV1-11 IGKV3-20, IGKV6-21, IGKV1-22, IGKV2-23, IGKV2-24, IGKV3-25, IGKV2-26, IGKV1-27, IGKV2-28, IGKV2-29, IGKV2-30, IGKV3-31, IGKV1-32, IGKV1-33, IGKV3-34, IGKV1-35, IGKV2-36, IGKV1-37 and all intergenic spacer sequences).

[0251] The constructed fusion BAC and Cre were electroporated into the positive cell 1A5-2A3-1G4-1C1 clone in the fourth step: the BAC also carried Neo resistance (Neo antibody had inverted terminal repeat sequences at both ends), and the human sequence on the BAC had loxP and lox2272 elements at both ends in the same direction as the positive clone in the fourth step, respectively. There was PB3' (3'ITR, 3'inverted terminal repeat, 3' inverted terminal repeat) between the lox2272 element and the human genomic sequence. Under the action of Cre recombinase, the sequences between loxP and lox2272 in the BAC and the positive cell clone in the fourth step were replaced, and Puro resistance was replaced by the human genomic sequence and Neo resistance on the BAC; the cells were screened with G418 drug, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0252] Sequence substitutions were identified using 10 pairs of primers (sequences are shown in Table 27 below, and primer identification positions are also marked on the map (Figure 58)). Two pairs of primers, VT1 and VT2, were used to identify random insertions, and only when the identification was negative could the cell be confirmed as a correct clone.

[0253] The remaining eight primer pairs simultaneously amplified four clones that produced positive bands ( FIG. 59 ): 1A5-2A3-1G4-1C1-2A5, 1A5-2A3-1G4-1C1-2A6, 1A5-2A3-1G4-1C1-2C7, and 1A5-2A3-1G4-1C1-2C8. These four clones were used as positive clones in the fifth step.

[0254] Table 27

[0255] 6. Electroporate pBase (PiggyBac transposase) into the cell clones obtained in step 5.

[0256] Electroporate pBase (PiggyBac transposase) into the positive cell clone 1A5-2A3-1G4-1C1-2C7 from step 5. Under the action of PiggyBac transposase, the two inverted terminal repeat sequences will be cut out from the genome, the Neo sequence and lox2272 will be lost, and the positive cell clone will only have the inserted human gene sequence and a loxP element. Select ES clones for culture and amplification, and then perform PCR typing and identification.

[0257] The sequence deletion was identified by three pairs of primers (sequences are shown in Table 28 below, and the primer identification positions are also marked on the map (Figure 60)).

[0258] There were 11 clones that simultaneously amplified positive bands using the three primer pairs ( Figure 61 ): 1A5-2A3-1G4-1C1-2C7-3A1, 1A5-2A3-1G4-1C1-2C7-3A2, 1A5-2A3-1G4-1C1-2C7-3A3, 1A5-2A3-1G4-1C1-2C7-3A5, 1A5-2A3-1G4-1C1-2C7-3A6, and 1A5-2A3- 1G4-1C1-2C7-3B2, 1A5-2A3-1G4-1C1-2C7-3B3, 1A5-2A3-1G4-1C1-2C7-3B4, 1A5-2A3-1G4-1C1-2C7-3B5, 1A5-2A3-1G4-1C1-2C7-3B6, and 1A5-2A3-1G4-1C1-2C7-3B8. These 11 clones were used as positive clones in the sixth step.

[0259] Table 28

[0260] 7. F0 mice obtained from ES1

[0261] The positive cell clones obtained in step 6 are injected into blastocysts, which are then transplanted into surrogate mothers. After a gestation period of about 20 days, F0 mice are born. The paws of 5-7 day old mice are cut, DNA is extracted, and PCR typing is performed to confirm the mouse genotype.

[0262] Six mice were born after injection of the 1A5-2A3-1G4-1C1-2C7-3B4 clone. All six mice were positive for the gene using eight primer pairs (Table 29). The genome of the F0 mice is shown in Figure 62 , and the results of the gene identification are shown in Figure 63 .

[0263] Table 29

[0264] 8. F1 mice obtained from ES1

[0265] The F0 mice identified as positive were mated with wild-type mice to obtain F1 generation mice.

[0266] The genotype of the mice was confirmed by PCR typing of the genomic DNA of the F1 generation mouse tail.

[0267] A total of six F1 mice were born. ES1 was identified using eight primer pairs (Table 30), and three mice (1#, 2#, and 6#) were positive. The genome of the F1 mice is shown in Figure 64, and the identification results are shown in Figure 65.

[0268] Table 30

[0269] ES Cell Line II

[0270] It contains approximately 20kb of human gene sequences (including IGKV2D-40, IGKV1D-39, IGKV2D-38 and all gene intergenic sequences) inserted between the mouse J region and C region.

[0271] The constructed vector is electroporated into wild-type ES cells. The vector carries Neo resistance. The cells are screened with G418 drug. The relevant ES clones are selected for culture and amplification, and then PCR typing is performed for identification:

[0272] The sequences were inserted into the corresponding positions using three pairs of primers (sequences are shown in Table 31 below, and the primer identification positions are also marked on the map (Figure 66)).

[0273] There were four clones that simultaneously amplified positive bands using the three pairs of primers ( FIG. 67 ): 1A4, 1A8, 2B1, and 2B4. These four clones were regarded as positive clones in the first step.

[0274] Table 31

[0275] 2. Approximately 20kb of human V genome sequence (including IGKV1D-37, IGKV2D-36, IGKV1D-35 and all intergenic sequences) was introduced with two lox sites loxp-lox5171 to obtain positive clones.

[0276] The constructed vector was electroporated into the positive cell 1A4 clone from the first step: the vector carries Puro resistance, and one end of the homology arm is set on the human sequence of the positive clone from the first step, so Neo resistance will be lost due to homologous recombination in this step, and the Puro resistance ends carry PB3' (3'ITR, 3'inverted terminal repeat, 3' reverse terminal repeat)-lox5171 and loxP elements respectively; the cells were screened with Puromycin drug, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0277] The sequences were inserted into the corresponding positions using three pairs of primers (sequences are shown in Table 32 below, and the primer identification positions are also marked on the map (Figure 68)).

[0278] There were five clones that simultaneously amplified positive bands using the three pairs of primers ( FIG. 69 ): 1A4-1D3, 1A4-1D5, 1A4-1D6, 1A4-1E1, and 1A4-1E5. These five clones were used as positive clones in the second step.

[0279] Table 32

[0280] 3. Insert about 350kb of human V genome sequence (including IGKV3D-34, IGKV1D-33, IGKV1D-32, IGKV3D-31, IGKV2D-30, IGKV2D-29, IGKV2D-28, IGKV1D-27, IGKV2D-26, IGKV3D-25, IGKV2D-24, IGKV2D-23, IGKV1D-22, I GKV6D-21, IGKV3D-20, IGKV2D-19, IGKV2D-18, IGKV6D-41, IGKV1D-17, IGKV1D-16, IGKV3D-15, IGKV2D-14, IGKV1D-13, IGKV1D-12, IGKV3D-11, IGKV2D-10, IGKV1D-42, IGKV1D-43, IGKV1D-8, IGKV3D-7, and all intergenic spacer sequences).

[0281] The constructed fusion BAC and Cre were electroporated into the positive cell 1A4-1E5 clone in the second step: the BAC also carried Neo resistance (Neo resistance had inverted terminal repeat sequences at both ends), and the human sequence on the BAC had loxP and lox5171 elements at both ends in the same direction as the positive clone in the second step, and there was PB5' (5'ITR, 5'inverted terminal repeat, 5' inverted terminal repeat) between the lox5171 element and the human genomic sequence. Under the action of Cre recombinase, the sequences between loxP and lox5171 in the BAC and the positive cell clone in the second step were replaced, and Puro resistance was replaced by the human genomic sequence and Neo resistance on the BAC; the cells were screened with G418 drugs, and the relevant ES clones were selected for culture, amplification, and PCR typing identification.

[0282] Sequence substitutions were identified using 10 pairs of primers (sequences are shown in Table 33 below, and primer identification positions are also marked on the spectrum in Figure 70). Two pairs of primers, VT1 and VT2, were used to identify random insertions, and only when the identification was negative could the cell be confirmed as a correct clone.

[0283] The remaining eight primer pairs simultaneously amplified seven clones that produced positive bands ( FIG. 71 ): 1A4-1E5-2C2, 1A4-1E5-2C4, 1A4-1E5-2C6, 1A4-1E5-2C7, 1A4-1E5-2F2, 1A4-1E5-2F5, and 1A4-1E5-2F7. These seven clones were used as positive clones in the third step.

[0284] Table 33

[0285] 4. Electroporate pBase (PiggyBac transposase) into the cell clones obtained in step 3.

[0286] pBase (PiggyBac transposase) is electroporated into the positive cell 1A4-1E5-2F5 clone from step 3. Under the action of PiggyBac transposase, the two inverted terminal repeat sequences will be cut out from the genome, the Neo sequence and lox5171 will be lost, and the positive cell clone will only carry the inserted human gene sequence and a loxP element. ES clones are selected for culture, amplification, and PCR typing.

[0287] The sequence deletion was identified by three pairs of primers (sequences are shown in 34 below, and the primer identification positions are also marked on the map (Figure 72)).

[0288] There were 7 clones that simultaneously amplified positive bands using the three pairs of primers (Figure 73): 1A4-1E5-2F5-1G1, 1A4-1E5-2F5-1G6, 1A4-1E5-2F5-1G7, 1A4-1E5-2F5-2F1, 1A4-1E5-2F5-2F2, 1A4-1E5-2F5-2F6, and 1A4-1E5-2F5-2F7. These 7 clones were used as positive clones in the third step.

[0289] Table 34

[0290] 5. F0 mice obtained from ES2

[0291] The positive cell clones obtained in the fourth step are injected into blastocysts, which are then transplanted into surrogate mother mice. After a gestation period of about 20 days, mice are born. The paws of 5-7 day old mice are cut, DNA is extracted, and PCR typing is performed to confirm the mouse genotype.

[0292] Six mice were born after injection of the 1A4-1E5-2F5-1G1 clone. All six mice were positive for the gene using six primer pairs (Table 35). The genome of the F0 mice is shown in Figure 74 , and the results of the gene identification are shown in Figure 75 .

[0293] Table 35

[0294] 6. F1 mice obtained from ES2

[0295] The F0 mice identified as positive were mated with CMV-Cre mice to obtain F1 mice.

[0296] The genotype of the mice was confirmed by PCR typing of the genomic DNA of the F1 generation mouse tail.

[0297] A total of six F1 mice were born. ES2 was identified using six primer pairs (Table 36), and CMV-Cre was identified using one primer pair. Two mice (2# and 4#) were identified as double-gene positive. The genome diagram of the F1 mice is shown in Figure 76, and the identification results are shown in Figure 77.

[0298] Table 36

[0299] 3. Hybridization of ES1 and ES2 mice

[0300] 1. ES1-positive F1 mice were crossed with double-gene F1-positive mice obtained from ES2 to obtain Cre-positive, ES1-positive and ES2-positive F2 mice.

[0301] Three pairs of primers (Table 37) were used to identify translocation of the gene, and one pair of Cre primers was used to identify Cre positivity.

[0302] Two mice were identified as ectopic and carrying Cre: 4# and 5#. Figures 78 and 79 show the schematic diagram of genomic hybridization and the identification results, respectively.

[0303] Table 37

[0304] 2. The obtained F2-positive mice were mated with wild-type mice to obtain 3.5-day blastocysts and establish ES cell lines. Different ES cell lines were tested by PCR to screen out cells that did not carry Cre but had undergone ectopic reprogramming. These cells contained human genomic sequences of ES1 and ES2 and had a loxP sequence between IGKV1-37 and IGKV3D-7.

[0305] Three pairs of primers (Table 38) were used to identify translocation of the gene, and one pair of Cre primers was used to identify Cre positivity.

[0306] Three clones were identified as ectopic and not carrying Cre: 1C2, 2D1, and 2D2. These three clones were designated as ES cell line III. Figures 80 and 81 show the schematic diagram of genomic hybridization and the identification results, respectively.

[0307] Table 38

[0308] 3. Perform a second homologous recombination KO on ES cell line III (knockout of all gene sequences from Igkj5 to Igkv2-137).

[0309] The constructed vector was electroporated into the positive cell clone 2D1 in the first step. The vector has Neo resistance. The cells were screened with G418 drug, and relevant ES clones were selected for culture and amplification, and then PCR typing and identification were performed.

[0310] Two pairs of primers (sequences shown in Table 39 below, with primer identification positions also noted on the map ( FIG. 82 )) identified that the genes between Igkj5 and Igkv2-137 had been recombined by Neo-resistance, indicating that a recombinant deletion had occurred in the genes between Igkj5 and Igkv2-137 of the mouse IgK chain.

[0311] There were 11 clones that simultaneously amplified positive bands using the two pairs of primers ( Figure 83 ): 2D1-1F1, 2D1-1F2, 2D1-1F3, 2D1-1F6, 2D1-1F7, 2D1-1H1, 2D1-1H2, 2D1-1H3, 2D1-1H5, 2D1-1H7, and 2D1-1H8. These 11 clones were used as positive clones in the second step.

[0312] Table 39

[0313] 4. Inject the positive cell clones obtained in the third step to obtain positive F0 mice

[0314] The positive cell clones obtained in the third step are injected into blastocysts, which are then transplanted into surrogate mother mice. After a gestation period of about 20 days, mice are born. The paws of 5-7 day old mice are cut, DNA is extracted, and PCR typing is performed to confirm the mouse genotype.

[0315] After the injection of clone 2D1-1F1, a total of 6 mice were born. All 6 mice were positive after identification using one pair of primers (Table 40). The identification results are shown in Figure 84.

[0316] Table 40

[0317] 5. Obtaining Positive F1 Mice

[0318] The F0 mice identified as positive were mated with wild-type mice to obtain F1 mice.

[0319] The genotype of the mice was confirmed by PCR typing of the genomic DNA of the F1 generation mouse tail.

[0320] A total of 6 F1 mice were born, and four mice (1#, 2#, 3#, and 6#) were positive after identification using two pairs of primers (Table 41). The identification results are shown in Figure 85.

[0321] Table 41

[0322] 6. Obtaining Homozygous Mice

[0323] Homozygous mice were obtained by mating F1 mice that were positive.

[0324] The genotype of the F2 generation mice was confirmed by PCR typing of the genomic DNA of the mouse tail.

[0325] A total of five F2 mice were born. Using three primer pairs (Table 42), identification confirmed that mouse #3 was homozygous; mice #2 and #5 were heterozygous; and mice #1 and #4 were wild-type. The identification results are shown in Figure 86.

[0326] Table 42

[0327] Breeding performance testing

[0328] The resulting homozygous mice were further bred using a one-male-one-female pairing. The reproductive performance of the mice was analyzed: a total of 24 breeding pairs were bred, and the number of mice born in each breeding pair one month after mating was counted. The results are shown in Table 43.

[0329] Table 43

[0330] There were a total of 24 breeding pairs, and 20 breeding pairs gave birth to mice one month after mating, with the effective breeding pairs reaching 87.5%. At the same time, the average litter size of mice was 5, which was consistent with the litter size of wild-type mice, confirming that the breeding performance of this genotype of mice was normal.

[0331] 5. Kappa chain (K chain) immunohistochemistry sequencing

[0332] The homozygous mice were euthanized and their spleens were dissected for RNA extraction. After the total RNA of the samples passed the test, libraries were constructed and then immune repertoire sequencing was performed (immune repertoire sequencing (Immuno-Seq) targets B / T lymphocytes and uses 5'RACE or multiplex PCR technology to specifically amplify the variable regions (V regions) that determine the diversity of B cell receptors (BCR) or T cell receptors (TCR), combined with high-throughput sequencing technology to comprehensively assess the diversity of the immune system). The sequenced sequences were quality controlled using quality control software and the sequencing background was filtered. They were then compared with the V and J genes of the IMGT immune cell receptor library to search for corresponding gene fragments, find the precise sites of V and J gene fragments and sequences, and statistically analyze information such as V and J gene frequencies, clone frequency distribution, and number of polypeptide sequences.

[0333] Based on the obtained gene usage frequency data, we plotted the gene usage frequency distribution at the read level. The results are shown in Figures 87 and 88. Based on the sequencing results, IGKV4-1 was confirmed to have the highest usage frequency at the read level in spleen RNA from unimmunized mice, followed by IGKV1-33 and IGKV3D-20, with usage frequencies decreasing in this order as shown in the figure.

[0334] Based on the sequencing results, it was confirmed that IGKJ1 had the highest usage frequency at the read level in the spleen RNA of non-immunized mice, followed by IGKJ4, IGJK3, IGKJ5, and IGKJ2 in descending order.

[0335] CDR3 amino acid sequence feature statistics are an important step in immune group analysis. By refining each clone, the sequence abundance of different CDR3 sequences evolved from each germline antibody sequence is quantitatively analyzed, and finally the sequence is conveniently selected for further antibody expression.

[0336] The Weblogo feature analysis of the CDR3 amino acid sequences of each sample at the read level is shown in Figure 89. The results show the amino acid sequence with the highest abundance in the CDR3 of unimmunized homozygous mice.

[0337] Example 3. Preparation and identification of mice with both heavy chain and kappa light chain genetic modifications

[0338] 1. Heavy chain homozygous mice and kappa light chain homozygous mice were bred at a 1:1 ratio. There was no gender restriction; the kappa light chain homozygous mice could be either female or male. One month after the breeding, mice were gradually born, and all born were digene heterozygous mice. The identification results are shown in Figures 90 to 93.

[0339] Twelve mice were born, and the heavy chain was identified by two pairs of primers, H-MT and H-WT (Table 44), and all the mice were heterozygous. The kappa light chain was identified by two pairs of primers, K-MT and K-WT, and also heterozygous mice, indicating that the 12 mice were double-gene heterozygous mice for the heavy chain and kappa light chain.

[0340] Table 44

[0341] 2. One male and two female mice heterozygous for both heavy and kappa light chains were mated. One month after mating, mice were born. When the offspring were 5-7 days old, their paws were clipped for genotyping. The results are shown in Figures 94 to 97.

[0342] Nineteen mice were born. The heavy chain was identified using two primer pairs, H-MT and H-WT, and the kappa light chain was identified using two primer pairs, K-MT and K-WT. In addition, two internal reference primers, F3-R3 and F6-R6, were added for each assay. The primer pairs are shown in Table 45.

[0343] The results showed that mouse No. 34 was a double-gene homozygous mouse.

[0344] Table 45

[0345] 3. The proportion of digene homozygous mice in the breeding pairs was counted. The results are shown in Table 46 below. The results show that the ratio of digene homozygous mice born is 1 / 16, which is consistent with Mendel's ratio.

[0346] Table 46

Claims

1. A method for preparing a genetically modified mouse, comprising: (i) obtaining a heavy chain gene locus modified mouse, comprising (ia) inserting a first partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the first mouse, wherein the first partial segment includes a first partial hIgHV continuous segment, the entire hIgHD segment, and the entire hIgHJ segment, the first partial segment does not include a segment between the downstream of the hIgHV1-2 gene and the upstream of the hIgHV6-1 gene, and includes a first recombination site upstream of the first partial segment and downstream of the mIgHJ region; (ib) inserting a second partial segment of a human immunoglobulin heavy chain variable region locus between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of a second mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment includes a second partial hIgHV continuous segment, and includes a second recombination site between the downstream of the second partial hIgHV continuous segment and the upstream of the mIgHC region; (ic) hybridizing the first mouse with the second mouse, and screening to obtain a third mouse, wherein the second partial segment of the human immunoglobulin heavy chain variable region locus and the first partial segment located downstream of the second partial segment are inserted between the mIgHJ region and the mIgHC region of the immunoglobulin heavy chain locus of the third mouse, and a third recombination site is present between the second partial segment and the first partial segment; and (id) knocking out the mIgHV5-1 gene and all upstream mIgHV segments of the immunoglobulin heavy chain locus, the continuous segment between the mIgHD1-1 gene and the mIgHJ4 gene, and the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes of the third mouse, respectively, to obtain the heavy chain locus gene-modified mice; (ii) obtaining a Kappa light chain gene locus modified mouse, wherein the immunoglobulin Kappa light chain gene locus of the Kappa light chain modified mouse includes all hIgKV segments, all hIgKJ segments, and mIgKC segments in sequence, and all mIgKV segments and all mIgKJ segments are knocked out; as well as (iii) breeding the heavy chain locus modified mice obtained in step (i) and the kappa light chain locus modified mice obtained in step (ii) to screen for double-positive mice, and interbreeding the obtained double-positive mice to screen for homozygous mice.

2. The method according to claim 1, wherein step (ii) comprises: (iia) inserting a first partial segment of a human immunoglobulin kappa light chain variable region locus between the mIgKJ region and the mIgKC region of the immunoglobulin kappa light chain locus of a fourth mouse, wherein the first partial segment includes a first partial hIgKV continuous segment and the entire hIgKJ segment, and includes a fourth recombination site upstream of the first partial segment and downstream of the mIgKJ region; (iib) inserting a second partial segment of the human immunoglobulin Kappa light chain variable region locus between the mIgKJ region and the mIgKC region of the immunoglobulin Kappa light chain locus of the fifth mouse, wherein the second partial segment is located upstream of the first partial segment, the second partial segment includes a second partial hIgKV continuous segment, the first partial hIgKV continuous segment and the second partial hIgKV continuous segment together constitute the entire hIgKV segment, and include a fifth recombination site between the downstream of the second partial hIgKV continuous segment and the upstream of the mIgKC region; (iic) hybridizing the fourth mouse with the fifth mouse to obtain a sixth mouse through screening, wherein the second partial segment of the human immunoglobulin Kappa light chain variable region locus and the first partial segment located downstream of the second partial segment are inserted between the mIgKJ region and the mIgKC region of the immunoglobulin Kappa light chain locus of the sixth mouse, and the sixth mouse has a sixth recombination site between the second partial segment and the first partial segment; (iid) Knock out all the mIgKV segments and all the mIgKJ segments of the immunoglobulin Kappa light chain locus of the sixth mouse to obtain the Kappa light chain locus modified mouse.

3. The method according to claim 2, having one or more of the following features: (a) the first part of the hIgKV continuous segment of the first part includes the continuous segment between the hIgKV4-1 gene and the hIgKV1-37 gene; (b) the second part of the hIgKV continuous segment of the second part includes the continuous segment between the hIgKV3D-7 gene and the hIgKV2D-40 gene; and (c) The fourth, fifth and sixth recombination sites are loxP sites.

4. The method of claim 2, wherein in step (iia), the first partial hIgKV continuous segment and the entire hIgKJ segment of the first partial segment are inserted between the mIgKJ region and the mIgKC region by at least two steps, the at least two steps comprising: (iia1) inserting a first continuous segment comprising hIgKV1-5 to hIgKJ5 genes between the mIgKJ region and the mIgKC region; and (iia2) inserting a second continuous segment comprising hIgKV1-37 to hIgKV1-6 genes upstream of the first continuous segment.

5. The method according to claim 4, wherein the step (iia1) specifically comprises: (iia11) inserting a continuous segment comprising hIgKJ1 to hIgKJ5 genes between the mIgKJ region and the mIgKC region; (iia12) inserting a continuous segment comprising hIgKV4-1 to hIgKV5-2 genes upstream of the inserted segment in step (iia11); (iia13) inserting a continuous segment gene comprising the upstream of hIgKV5-2 to the downstream of hIgHKV2-4 upstream of the insertion segment of step (iia12); and (iia14) Insert the continuous segment between hIgHKV2-4 and hIgKV1-5 genes and loxP and lox2272-PB5' sites upstream of the inserted segment in step (iia13).

6. The method according to claim 5, wherein the step (iia2) specifically comprises: (iia21) recombining the genome obtained in step (iia1) with a BAC vector comprising a second continuous segment between hIgKV1-6 and hIgKV1-37 genes, loxP and lox2272 sites located at both ends of the second continuous segment in the same orientation as in step (iia14), and Cre recombinase, wherein PB3' is included between the lox2272 site and the second continuous segment; (iia22) contacting the recombinant genome obtained in step (iia21) with PiggyBac transposase to screen for a genome comprising the first partial segment and the loxP site located upstream of the first partial segment.

7. The method according to any one of claims 2 to 6, wherein in step (iib), the second partial hIgKV continuous segment of the second partial segment is inserted upstream of the first partial segment by at least the following steps, comprising: (iib1) inserting a continuous segment comprising hIgKV2D-40 to hIgKV2D-38 genes between the mIgKJ region and the mIgKC region; and (iib2) inserting the continuous segment between hIgKV1D-37 and hIgKV1D-35 genes and the PB3'-lox5171 and loxP sites downstream of the inserted segment in step (iib1); (iib3) recombining the genome obtained in step (iib2) with a BAC vector containing the continuous segment between hIgKV3D-34 and hIgKV3D-7 genes and loxP and lox5171 located at both ends in the same direction as in step (iib2), and Cre recombinase, wherein PB5' is contained between the lox5171 site and the continuous segment described in this step; (iib4) contacting the recombinant genome obtained in step (iib3) with PiggyBac transposase to screen for a genome comprising the second partial segment and the loxP site located downstream of the second partial segment.

8. The method of claim 7, wherein step (iic) comprises: (iic1) screening Cre-positive mice containing the first and second segments; (iic2) mating the mice obtained in step (iic1) with wild-type mice, and screening mice that do not carry Cre but contain the first partial segment and the second partial segment, wherein there is a loxP site between the second partial segment and the first partial segment.

9. The method of claim 2, wherein step (iid) comprises: (iid1) mating the sixth mouse with wild-type mice to screen positive mice; (iid2) The positive mice obtained in step (iid1) are mated between males and females to screen for homozygous mice.

10. The method according to claim 1, having one or more of the following features: (a) The first part of the hIgHV continuous segment of the first part includes the continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (b) the second part of the hIgHV continuous segment of the second part includes the continuous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; and (c) The first, second and third recombination sites are loxP sites.

11. The method according to claim 1 or 10, wherein the immunoglobulin heavy chain locus of the genetically modified mouse comprises: (i) Mouse Adam6a gene; (ii) Mouse Adam6b gene; (iii) The continuous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (iv) The continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene; (v) The continuous segment between the hIgHV6-1 gene and the hIgHJ6 gene; (vi) The mIgHC region.

12. The method of claim 1 , wherein in step (ia), the first partial hlgHV continuous segment, the entire hlgHD segment, and the entire hlgHJ segment of the first partial segment are inserted between the mIgHJ region and the mIgHC region by at least two steps, the at least two steps comprising: (ia1) inserting the first continuous segment comprising hIgHV6-1 to hIgHJ6 genes between the mIgHJ region and the mIgHC region; and (ia2) inserting a second continuous segment comprising hIgHV4-28 to hIgHV1-2 genes upstream of the first continuous segment.

13. The method according to claim 12, wherein the step (ia1) specifically comprises: (ia11) inserting a continuous segment comprising hIgHD6-25 to hIgHJ6 genes between the mIgHJ region and the mIgHC region; (ia12) inserting a continuous segment comprising hIgHD6-13 to hIgHD5-24 genes upstream of the inserted segment in step (ia11); (ia13) inserting a continuous segment comprising hIgHD1-1 to hIgHD5-12 genes upstream of the inserted segment in step (ia12); and (ia14) Insert the continuous segment between the hIgHV6-1 and hIgHD1-1 genes upstream and the loxP and lox2272-PB5' sites upstream of the inserted segment in step (ia13).

14. The method according to claim 13, wherein the step (ia2) specifically comprises: (ia21) recombining a BAC vector comprising a second continuous segment between hIgHV4-28 and hIgHV1-2 genes, loxP and lox2272 sites located at both ends of the second continuous segment in the same direction as in step (ia14), and Cre recombinase with the genome obtained in step (ia1), wherein PB3' is contained between the lox2272 site and the second continuous segment; (ia22) contacting the recombinant genome obtained in step (ia21) with PiggyBac transposase to screen for a genome comprising the first partial segment and the loxP site located upstream of the first partial segment.

15. The method according to any one of claims 10 to 14, wherein in step (ib), the second partial hIgHV continuous segment of the second partial segment is inserted upstream of the first partial segment by at least the following steps, comprising: (ib1) inserting a continuous segment comprising hIgHV3-74 to hIgHV3-72 genes between the mIgHJ region and the mIgHC region; and (ib2) inserting the continuous segment between hIgHV2-70 and hIgHV1-69D genes and the PB3'-lox5171 and loxP sites downstream of the inserted segment in step (ib1); (ib3) recombining the genome obtained in step (ib2) with a BAC vector comprising a continuous segment between hIgHV1-69-2 and hIgHV3-30 genes and loxP and lox5171 located at both ends in the same direction as in step (ib2), and Cre recombinase, wherein PB5' is contained between the lox5171 site and the continuous segment described in this step; (ib4) contacting the recombinant genome obtained in step (ib3) with PiggyBac transposase to screen for a genome comprising the second partial segment and the loxP site located downstream of the second partial segment.

16. The method of claim 15, wherein step (ic) comprises: (ic1) screening Cre-positive mice containing the first and second segments; (ic2) mating the mice obtained in step (ic1) with wild-type mice, and screening mice that do not carry Cre but contain the first partial segment and the second partial segment, wherein there is a loxP site between the second partial segment and the first partial segment.

17. The method of claim 1, wherein step (id) comprises: (id1) mating the third mouse with wild-type mice to screen positive mice; (id2) The positive mice obtained in step (id1) are mated between males and females to screen for homozygous mice.

18. A genetically modified mouse genome, wherein The immunoglobulin heavy chain loci of the genetically modified mouse include: (i) mouse Adam6a gene; (ii) mouse Adam6b gene; (iii) a second partial segment of the human immunoglobulin heavy chain variable region locus, which includes a second partial hIgHV continuous segment; (iv) a first partial segment of the human immunoglobulin heavy chain variable region locus, which is located downstream of the second partial segment and includes the first partial hIgHV continuous segment, all hIgHD segments and all hIgHJ segments, and the first partial segment does not include the segment between the downstream of the hIgHV1-2 gene and the upstream of the hIgHV6-1 gene; and (v) mIgHC region; and The immunoglobulin Kappa light chain locus of the genetically modified mouse includes, in sequence: (i) a second portion of a segment of a human immunoglobulin Kappa light chain variable region locus, which includes a second portion of a hIgKV continuous segment; (ii) a first partial segment of the human immunoglobulin Kappa light chain variable region locus, which is located downstream of the second partial segment and includes a first partial hIgKV continuous segment and the entire hIgHJ segment, the first partial segment and the second partial segment together constituting the entire hIgKV segment; and (iii) the mIgKC region.

19. The mouse genome of claim 18, having one or more of the following characteristics: (i) the second part of the human immunoglobulin heavy chain variable region locus hIgHV continuous segment includes or is a continuous segment between the hIgHV3-74 gene and the hIgHV3-30 gene; (ii) the first portion of the human immunoglobulin heavy chain variable region locus includes or is a continuous segment between the hIgHV4-28 gene and the hIgHV1-2 gene and a continuous segment between the downstream hIgHV6-1 gene and the hIgHJ6 gene; (iii) the immunoglobulin heavy chain locus of the mouse does not include the mIgHV5-1 gene and all upstream mIgHV segments, the continuous segment between the mIgHD1-1 gene and the mIgHJ4 gene, and the mIgHD3-1, mIgHD5-1, and mIgHD1-3 genes; (iv) the second hIgKV continuous segment includes or is the continuous segment between the hIgKV3D-7 gene and the hIgKV2D-40 gene; (v) the first hIgKV continuous segment includes or is the continuous segment between the hIgK1-37 gene and the hIgKJ5 gene; (vi) the fertility of the mice is not reduced compared to non-genetically modified wild-type mice; (vii) the first partial segment and the second partial segment of the heavy chain and the kappa light chain are not rearranged; and (viii) The mouse is capable of producing human-mouse chimeric antibodies, wherein the chimeric antibodies comprise a human heavy chain variable region and a mouse heavy chain constant region, and a human kappa light chain variable region and a mouse kappa light chain constant region.

20. A cell, tissue, organ or mouse comprising the mouse genome of claim 18 or 19; preferably, the cell is an embryonic cell, a B cell or a hybridoma cell; preferably, the tissue is the white pulp of the spleen or its lymph nodes; preferably, the organ is the spleen.

21. A method for preparing a monoclonal antibody, comprising: (a) immunizing a mouse having the genome of claim 18 or 19 with an antigen; (b) isolating cells producing monoclonal antibodies against the antigen from the mouse; and (c) culturing the cells to obtain the monoclonal antibody; Preferably, the cell is a spleen cell, a B cell or a hybridoma cell; preferably, the monoclonal antibody has a human heavy chain variable region, a human kappa light chain variable region, a mouse heavy chain constant region and a mouse kappa light chain constant region, and does not have a mouse heavy chain variable region and a mouse kappa light chain variable region.

22. Use of the cell, tissue, organ or mouse according to claim 20 in preparing a monoclonal antibody; preferably, the monoclonal antibody has a human heavy chain variable region and a human kappa light chain variable region, and does not have a mouse heavy chain variable region and a mouse kappa light chain variable region; preferably, the monoclonal antibody has a human heavy chain variable region, a human kappa light chain variable region, a mouse heavy chain constant region and a mouse kappa light chain constant region.

Images