Establishment method and application of transgenic mouse

Abstract

The invention provides an establishment method of a transgenic mouse, and the method comprises the following steps: a transgenic mouse MHC chimeric gene is introduced into an endogenous B2m gene locus of the mouse, the MHC chimeric gene comprises a human B2M gene and a chimeric alpha-chain gene, and the chimeric alpha-chain gene comprises a human alpha1 region, a human alpha2 region and a mouse alpha3 region; the chimeric alpha-chain gene further comprises a mouse transmembrane region and a mouse intracellular region. The transgenic mouse disclosed by the invention can be applied to tumor specific antibody detection, in-vivo evaluation of vaccines, and in-vivo screening of TCR-T and other drugs.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a method for establishing transgenic mice using gene editing technology and its application. Background Art

[0002] Tumor vaccines utilize tumor cell-associated antigens to activate the body's immune system against cancer. Their goal is to prevent and treat cancer, or to prevent its recurrence. The mechanism of action for tumor vaccines is as follows: Antigens are injected subcutaneously or intramuscularly into the body and taken up by dendritic cells (DCs). DCs home to lymph nodes via lymphatic vessels, where they are home to large numbers of naive T cells. These naive T cells bind to MHC class I / II molecules on the surface of antigen-presenting cells (APCs), becoming activated into CD8- or CD4-positive T cells. These activated T cells then circulate through the bloodstream and reach the tumor site, where they kill the tumor cells.

[0003] For tumor vaccines to be effective in the human body, they rely on the antigen presentation process of human MHC. Mice are the most important model for tumor vaccine development. However, mouse MHC presents similar, but not identical, epitopes to human MHC. Therefore, using traditional mouse MHC models to study the effects of recognition of human MHC (HLA)-restricted epitopes on T cell activation and subsequent in vivo effects presents challenges. To improve such studies, scientists have generated humanized mice expressing human leukocyte antigens (HLA). These mice can present human HLA-restricted epitopes to T cells, mimicking human immunity, thus narrowing the gap between mice and humans and offering broad application prospects in tumor vaccine development.

[0004] Previous data have shown that human transgenic HLA-A2 expression levels are low in HLA-humanized mice. Furthermore, even the latest third-generation HLA transgenic mice (HHDII), which utilize mouse-derived α3, transmembrane, and intracellular regions, exhibit significantly lower peripheral blood CD8+ T cell counts than normal mice. For example, homozygous B-HLA-A2.1 mice exhibit significantly reduced CD8+ T cell percentages and significantly increased CD4+ T cell and NK cell percentages, suggesting that the replacement of mouse B2m with hB2M-HLA-A2.1-H-2D in this protocol may affect CD8+ T cell development and, in turn, the proportions of splenic T cell subtypes. Summary of the Invention

[0005] The present disclosure provides a method for establishing transgenic mice, and the CD8+ T cells of the transgenic mice established can still be successfully activated, and HLA is highly expressed. The transgenic mice have human α1, α2 and mouse α3, transmembrane and intracellular regions. Human α1 and α2 can present antigens with human HLA antigen binding regions, and mouse α3, transmembrane and intracellular regions can bind to CD8 molecules, which helps T cells recognize antigens. Based on the product advantages, HLA humanized mouse cells can present and recognize peptide epitopes that are similar or identical to epitopes presented by humans, and can be used for tumor-specific antibody detection, in vivo evaluation of vaccines, and in vivo screening of drugs such as TCR-T. It has high application value in the fields of immunity and vaccine development.

[0006] According to one aspect of the present disclosure, a method for establishing a transgenic mouse is provided, the method comprising: introducing a transgenic mouse MHC chimeric gene into the mouse's endogenous B2m locus, the MHC chimeric gene comprising a human B2M gene and a chimeric α chain gene, the chimeric α chain gene comprising a human α1 region, a human α2 region and a mouse α3 region, the chimeric α chain gene also comprising a mouse transmembrane region and a mouse intracellular region.

[0007] In some embodiments, the cells of the transgenic mouse express human B2M protein and chimeric α chain.

[0008] In some embodiments, the expression of endogenous B2m protein in cells of the transgenic mouse is reduced or absent.

[0009] In some embodiments, the MHC chimeric gene replaces the nucleotide sequence of exon 2 to exon 3 of the endogenous mouse B2M gene.

[0010] In some embodiments, the chimeric α chain gene comprises nucleotide sequences encoding α1 and α2 of human HLA.

[0011] In some embodiments, the human HLA is selected from human HLA-A, human HLA-B, or human HLA-C.

[0012] In some embodiments, the human HLA-A includes HLA-A*02:01, HLA-A*24:02, HLA-A*01:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*03:01, HLA-A*11:01, HLA-A*23 :01, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A*31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*68:02 or HLA-A*69:01.

[0013] In some embodiments, the chimeric α chain gene further comprises nucleotide sequences encoding α3, transmembrane region, and intracellular region of the murine H2D protein.

[0014] In some embodiments, the human B2M gene comprises the nucleotide sequence of exon 2, intron 2, and exon 3 of human B2M.

[0015] In some embodiments, the human B2M gene and the chimeric α chain gene are connected via a linker sequence encoding a linker function.

[0016] In some embodiments, the MHC chimeric gene comprises the nucleotide sequence shown in SEQ ID NO: 30 or 36, or a nucleotide sequence having at least 90% sequence identity thereto, or a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides compared thereto.

[0017] In some embodiments, the transgenic mouse has the amino acid sequence shown in SEQ ID NO: 31 or 37.

[0018] In some embodiments, the transgenic mouse is a transgenic mouse.

[0019] In some embodiments, the method includes: using a recombinant vector of the B2M gene to replace exon 2 and exon 3 of the endogenous mouse B2M gene.

[0020] In some embodiments, the recombinant vector of the B2m gene comprises an MHC chimeric gene, a 3' homology arm, and a 5' homology arm.

[0021] In some embodiments, the 3' homology arm comprises the nucleotide sequence shown in SEQ ID NO:3.

[0022] In some embodiments, the 5' homology arm comprises the nucleotide sequence shown in SEQ ID NO:4.

[0023] In some embodiments, the method comprises the steps of: (1) Determine the target site based on the sequence of intron 1 and the intron between exons 3-4 of the mouse B2m gene; (2) Synthesizing the crRNA sequence according to the target site determined in step (1); (3) constructing a linear targeting vector containing the MHC chimeric gene; (4) introducing the crRNA obtained in step (2) and the linear targeting vector obtained in step (3) into mouse embryonic stem cells to obtain mouse embryonic stem cells that express human B2M protein and MHC chimeric α chain and have reduced or absent endogenous B2m gene expression; (5) The mouse embryonic stem cells obtained in step (4) are aggregated with tetraploid cells to prepare transgenic mice.

[0024] In some embodiments, in step (5), the transgenic mouse is prepared by tetraploid compensation technology.

[0025] In some embodiments, in step (2), the crRNA includes the sequence shown in SEQ ID NO: 1 and / or the sequence shown in SEQ ID NO: 2.

[0026] According to another aspect of the present disclosure, a tissue, body fluid, cell, or fragment thereof, or an extract thereof, of a transgenic mouse is provided. The transgenic mouse is constructed using the method described in the present disclosure or is a progeny thereof. The cells or tissues cannot develop into an animal individual, and the cells do not include germ cells.

[0027] In some embodiments, the genome of the cells or tissues of the transgenic mouse comprises a nucleotide sequence as shown in SEQ ID NO: 30 or 36, or a nucleotide sequence having at least 90% sequence identity thereto, or a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides compared thereto.

[0028] In some embodiments, the cells or tissues of the transgenic mouse include the amino acid sequence shown in SEQ ID NO: 31 or 37.

[0029] In some embodiments, the CD8+ T cell content of the transgenic mouse is comparable to the CD8+ T cell content of a wild-type mouse.

[0030] According to yet another aspect of the present disclosure, a chimeric gene is provided, comprising a human B2M gene and a chimeric α chain gene.

[0031] In some embodiments, the chimeric gene further comprises a nucleotide sequence encoding a linker protein having a connecting function.

[0032] In some embodiments, the chimeric gene comprises a nucleotide sequence as shown in SEQ ID NO: 30 or 36, or a nucleotide sequence having at least 90% sequence identity thereto, or a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides compared thereto.

[0033] In some embodiments, the chimeric gene encodes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 31 or 37.

[0034] According to another aspect of the present disclosure, a recombinant vector is provided, wherein the recombinant vector comprises the chimeric gene described in the present disclosure.

[0035] In some embodiments, the recombinant vector further comprises a 3' homology arm and a 5' homology arm, wherein the 3' homology arm is as shown in SEQ ID NO: 3, and the 5' homology arm is as shown in SEQ ID NO: 4.

[0036] According to another aspect of the present disclosure, a method for evaluating an intervention regimen is provided, the evaluation method comprising implanting tumor cells into an individual, applying the intervention regimen to the individual implanted with the tumor cells, and detecting and evaluating the tumor suppression effect on the individual after application of the intervention regimen; wherein the individual is selected from a transgenic mouse constructed by the method described in the present disclosure.

[0037] According to another aspect of the present disclosure, provided is the use of the method, the chimeric gene, the recombinant vector or the evaluation method described in the present disclosure in the development of products requiring immune processes involving human cells, the manufacture of human antibodies, vaccines, or as a model system for pharmacology, immunology, microbiology, and medical research.

[0038] According to another aspect of the present disclosure, provided is the application of the method, the chimeric gene, the recombinant vector or the evaluation method described in the present disclosure in producing and utilizing animal experimental disease models for human cell transplantation, immune system reconstruction, and pathogenesis research.

[0039] According to another aspect of the present disclosure, provided is the use of the method, the chimeric gene, the recombinant vector or the evaluation method according to the present disclosure in the formation and function study of human hematopoietic stem cells and / or construction of disease models.

[0040] According to another aspect of the present disclosure, provided is the application of the method, the chimeric gene, the recombinant vector or the evaluation method according to the present disclosure in screening, verifying, evaluating or studying MHC function, human MHC signaling mechanism, human-targeted antibodies, vaccines, human-targeted drugs, drug efficacy, immune-related disease drugs and anti-tumor or anti-inflammatory drugs, screening and evaluating human drugs and drug efficacy research. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 A schematic diagram of the targeting strategy for HLA-A*0201 humanized mice in the embodiments of the present disclosure is shown.

[0042] Figure 2Shown is a schematic diagram of the targeting vector of the HLA-A*0201 humanized mouse and its sequencing result information in the embodiments of the present disclosure.

[0043] Figure 3 A gel image showing the cell identification results of HLA-A*0201 humanized mice in the examples of the present disclosure is shown.

[0044] Figure 4 A gel image showing the cell identification results of HLA-A*0201 humanized mice in the examples of the present disclosure is shown.

[0045] Figure 5 The figure shows the stem cell sequencing result information of the HLA-A*0201 humanized mouse in the examples of the present disclosure.

[0046] Figure 6 Shown are the genotype identification results of the HLA-A*0201 humanized mice in the Examples of the present disclosure.

[0047] Figure 7 Shown are the FACS results of protein expression analysis in the blood of HLA-A*0201 humanized mice in the Examples of the present disclosure.

[0048] Figure 8 Shown are the FACS results of T cell ratio analysis in the blood of HLA-A*0201 humanized mice in the Examples of the present disclosure.

[0049] Figure 9 Shown are the genotype identification results of the HLA-A*2402 humanized mice in the Examples of the present disclosure.

[0050] Figure 10 Shown are the FACS results of protein expression analysis and T cell ratio analysis in the blood of HLA-A*2402 humanized mice in the Examples of the present disclosure. DETAILED DESCRIPTION

[0051] MHC is a collective term for a group of genes encoding mammalian major histocompatibility antigens. These genes are divided into MHC class I, MHC class II, and MHC class III, encoding MHC class I, MHC class II, and MHC class III molecules, respectively. The human MHC is also known as the human leukocyte antigen (HLA) complex. The mouse MHC is known as the H2 complex. "H2-K," "H2-D," and "H2-L" refer to the mouse MHC I α protein subclasses, all encoded on mouse chromosome 17. H2-D1 belongs to the mouse MHC class I molecule.

[0052] Human MHC class I molecules are heterodimers composed of an α heavy chain and a β2-microglobulin (β2m) light chain. Based on their cellular distribution, they can be divided into an extracellular domain, a transmembrane domain, and an intracellular domain. The amino terminus is located outside the cell membrane, while the carboxyl terminus resides in the cytoplasm. The extracellular domain of the α chain folds into three functional domains: the α1, α2, and α3 domains, according to different spatial conformations. The amino acid sequences of the α1 and α2 domains vary significantly, representing polymorphic sites. The α3 domain is relatively conserved, with a structure similar to that of immunoglobulin superfamily molecules. It is species-specific and serves as the site of CD8 binding. The β2m light chain (B2M) non-covalently binds to the α3 domain, attaching to a conserved immunoglobulin-like region. While not directly involved in antigen presentation by MHC class I molecules, it facilitates the transport of newly synthesized MHC class I molecules from the endoplasmic reticulum to the cell surface and plays a supporting role in the structural stability and cell surface expression of MHC class I molecules.

[0053] Class I HLA genes are located on the short arm of chromosome 6 and encode MHC-I proteins. Their heavy chains can be HLA-A, HLA-B, or HLA-C monomers, while their light chains are β-2-microglobulin. Class I HLA-A, HLA-B, or HLA-C molecules are highly polymorphic. The antigens / ligands for many of these molecules remain unknown, but they can interact with CD8+ T cells, NKT cells, and NK cells. HLA-A2 is the most common type at the HLA-A locus, accounting for approximately 50% of HLA-A2-positive individuals in the Han Chinese population. Other subtypes with higher frequency in the Chinese population include HLA-A*0201, HLA-A*0203, HLA-A*0206, and HLA-A*0207. These subtypes differ only in a few amino acids in the antigen-binding groove.

[0054] To study the pathogenic mechanisms associated with HLA, it is necessary to construct corresponding transgenic mouse models to provide experimental animal models for diseases such as tumors and inflammation. However, existing HLA humanized mice commonly suffer from technical bottlenecks such as insufficient HLA expression and significantly reduced CD8+ T cell counts.

[0055] To this end, the present disclosure provides an HLA-humanized mouse that replaces the mouse B2m segment with human HLA. By way of example, the present disclosure successfully generated an HLA-A*0201 humanized mouse by replacing the mouse B2m segment with HLA-A. Those skilled in the art will appreciate that other HLA subtypes, including but not limited to HLA-B and HLA-C, can also be used to achieve the corresponding objectives using the methods of the present disclosure.

[0056] In this study, C57BL6J mice were used as the research background. HLA-A*02:01, which is highly frequent in the human population, was selected to replace the mouse B2m segment. Specifically, using the HHD (HLA-A2.1 transgenic mouse) strategy, exons 2 and 3 of human B2M, human α1, human α2, and mouse α3, along with the mouse transmembrane region and intracellular region, were inserted into the mouse B2m gene locus through homologous recombination. The goal was to enable the mouse to utilize the expression regulation of the endogenous B2m gene and express a humanized B2M-HLA fusion protein. Human α1 and α2 can present antigens using the human HLA-A*0201 antigen-binding region, while the mouse α3, transmembrane region, and intracellular region can bind to CD8 molecules, facilitating antigen recognition by T cells. The results showed that HLA-A*0201 humanized mice were successfully prepared, with HLA-A2 and hB2M successfully expressed, and mB2m successfully deleted, making H2D b While no expression is observed, analysis of publicly available data indicates high levels of CD8+ T cell activation. Therefore, HLA-A*02:01 humanized mice can be used as a product for breeding and expansion. These transgenic mice can be used in applications such as tumor vaccine development and testing, in vivo screening and validation of TCR-T or immunotherapy drugs, and in immunology and vaccine development.

[0057] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0058] Definition Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0059] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0060] In this disclosure, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0061] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical, matched positions shared by the sequences over the comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where the same nucleotide or amino acid is present in both the target and reference sequences. Since gaps are not nucleotides or amino acids, gaps present in the target sequence are not counted. Similarly, since target sequence nucleotides or amino acids are counted and nucleotides or amino acids from the reference sequence are not counted, gaps present in the reference sequence are not counted.

[0062] Percent sequence identity can be calculated by determining the number of positions at which the identical amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. Comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using software that is readily available for online use and download. Suitable software programs are available from various sources for alignment of protein and nucleotide sequences. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available on the BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm to compare two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0063] In the present disclosure, the term "homologous arms" refers to flanking sequences on both sides of the exogenous sequence to be inserted into the targeting vector that are completely identical to the genomic sequence and are used for recognition and recombination.

[0064] In the present disclosure, the term "chimeric gene" includes chimeras of genes of the same or different species from the same or different sources.

[0065] In the present disclosure, the non-human animal is a mammal. Preferably, the non-human animal is a small mammal, such as a family of the family Jerboas. In one embodiment, the non-human animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the family Muridae. In one embodiment, the genetically modified animal is from a family selected from the family of the family Muridae (e.g., mouse-like hamsters), the family of the family Cricetidae (e.g., hamsters, New World rats and mice, voles), the family of the superfamily Murinoidea (true mice and rats, gerbils, spiny mice, crested rats), the family of the family Murinoidea (climbing mice, rock mice, tailed rats, Madagascar rats, and mice), the family of ... animal of the rodent. In one embodiment, the animal of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of the family of In a specific embodiment, the rodent is selected from the group consisting of a mouse and a rat. In one embodiment, the non-human animal is a mouse.

[0066] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0067] Example 1: Based on the mouse B2m gene sequence and transcript sequence (Gene ID: 12010), crRNA-1 that recognizes the 5' target site and crRNA-2 that recognizes the 3' target site were designed and synthesized. The 5' target site and the 3' target site are located in intron 1 and the intron between exons 3 and 4 of the B2m gene, respectively. The target sequences of each crRNA on B2m are as follows: 5' end crRNA-1: UAAUUUCUACUCUUGUAGAUCCGUUCUUCAGCAUUUGGAUUUC (SEQ ID NO: 1); PAM: TTTC; 3' end crRNA-2: UAAUUUCUACUCUUGUAGAUUUGACUUUAAAUAAUGGGCCUUU (SEQ ID NO: 2); PAM: TTTC; The crRNA sequence was synthesized by GenScript Biotech.

[0068] Example 2: HLA humanization strategy Targeting strategies for HLA humanization include Figure 1As shown, the mouse B2m leader peptide sequence and mouse intron 1 sequence were retained. The introns within the donor region containing the knockout region were all human sequences, with the WPRE added to the 3' end. The targeting vector consists of the following: 5' homology arm, hB2M-exon 2, hB2M-intron 2, hB2M-exon 3, HLA-α1, HLA-α2, H2D-α3, H2D-transmembrane region, H2D-intracellular region, WPRE, and 3' homology arm. The hB2M-exon 3 and HLA-α1 are connected by a linker (G4S)3.

[0069] Among them, HLA-A*02:01, which appears more frequently in the human population, was selected to replace the B2m fragment of the mouse. The model after targeting has human α1, α2 and mouse α3, transmembrane and intracellular regions. Human α1 and α2 can present antigens with the human HLA-A*0201 antigen binding region, and mouse α3, transmembrane and intracellular regions can bind to CD8 molecules, which helps T cells recognize antigens.

[0070] The sequences of the various elements of the targeting vector are shown in Table 1. Figure 2 shown.

[0071] Table 1 Component Sequence SEQ ID NO: 3 arm aggagctgtttatagacagctcaaacatgataagcatcactgtatgaaagacattaattagggtggcttgatcagctacagaaggatcctttggggtcacattcttttatcctgtggactggcaggaaggaggaacgtagccatgtcactggccctctaaagggagaacagcacctttgtgcatgcagcaaggcaggtaggcaggtaggcaggtaggcaggcgggtggtcagttacacagcctgcatgggtttgccgaccttctgttttaaccctgccagttctgctgagaaggcctgggacgatgagcttgctgtctttgtacagcagtggccttgctcctcacccagaggagccgagtgacagagctctgcgggtacatcttagccctttcagtctctagttggtaggcactagatttaccttctggaggcttccggacactcagggaaagaaatggacagggttgtaacttcatgtaaggcaccgtcactgatgtgtcagaaggaagttgaggagcgtgagagggaacgtgggtgtctctgtcaggtggagtctagtggtagaaaatccagcttttcggtgaaatccaTggcccttgaggccaaaagctcactcaaatcttgtattatatttatttctaggaaaggggaattgatgaagggggtggggatgggtgatctgcccagacaagcagttaccaaatggtggagccagaactcacctcctttaagaaggtgattgtcctacaaactcatttgatggtgaggtctggaatgtaaataatgtaatgttcggctaaccttctccttctctccctcccttccccttttcttttcagctctgaagattcatttgaacctgcttaattacaaatccagtttctaatatgctatacaatttatgcacgcagaaagaaatagcaatgtacacatcaccttctttatatcttactttaaatattttatgcatgttttcaaaaaattggaaatatcctagatagctgagcaataaatcttcaataagtattttgatcagaataataaatataattttaagaacaatagttgatcatatgccaaaccctctgtacttctcattacttggatgcagttactcatctttggtctatcacaacataagtgacatactttccttttggtaaagcaaagaggcctaattgaagtctgtcactgtgcccaatgcttagcaattctcacccccaaccctgtggctacttctgcttttgttacttttactaaaaataaaaaactaaaaaaaaaaaaaaaaattgttgccagacattgccagtgttacatgaagtggtttctaggtagcataaatgcctgcaagcacgtgcgtatttttcatcatctaaagaggcagtgggtatcttagaaggttcttgcagaagcaggctcctcaactaagggtcgagaggaaagtgactaggaaaggcctccaaagcagagcagtaggaggtggtgatgaaggtcaggga 3 5 arm caagtgtgttgagtcatgctgtgacctcagagctggggaggctgagacaggcgttcaaggccagcctggaagacagtcagactgtatctcaaattaaataataagcatataagtaaatgaaggggaaaacacaaaaagagccagtgaaatggctcagcaggtacaggcgcttaccactaagcctgagtgtatctcagatcccacttggtggacggagagaactgacttttaaatccacacatgagctacgtttcgtgcatgtgcacacacacacacacacacacacacacacacacacacacatatatactctctcagtaaaacaataatttttttaagaaagtagatatatacattagtgataccttggtatcagatctgcatattctacacagtagtactccttgtcatgttggttgagaagcagaaacagagttcaaagatggcagtggtatggctcagcagctaaaagtacctgccatgcaacctgaccacctgagttcatagaacccacgacagaaggaggagaaaactgtctcctgaaagccatccaatgtcttcacaagtacacctttgggaacttgactgtgtgtatgtgtgtgtgcacatacaaataacaatggaaagagtttacattattttctttccatgtccctatagcgtgcagtagctattcccaacatgttgagcgacttcagctatctgaaagccccatatccccttgcacacactgcttacttccagcagagtgcattctcaattgtcatggtcctcacatctccctctggtcctttacccatcccaactcagttgcttaaagctttcctagcttctgtggtattttaaattttgtttgttttctagttcatgaaaccagaggtttggtttcagaatgcaaactctgggctggaacagagaaaccctgggttgactcgcataggaacctcattagggaggagccaatgctaacacctgccacctgaggggtaattgctcagctctcagcactggatcagacatatgtgttgggaagtctagggaggagcaataagcaaaaacgaaaggggatgaaaataagaaagaaaagtgagaggggctgacacagcgcacaagcactgcgggctttgatgtggatactgtgaagggtgtgcagaatgggatgtgacgtttggaaagttggtgggatttattaagggaagggaggggcacagttcttgagagcttccaataataaaagctccttaaaaatccactgacagaagacactgctaaaagccaggtagggaaaacagaaggtactcgtaggattttgaggaaaagatactacgttttcaaaatgtgggtagactttgggggaagcagatcacttatccagagtagaaatggaacagggagaaatagaggaacaaatgtaagatggtgcacggtgcagactgagctctgttttcatctgtcttcccctgtggccctcag 4 hB2M - exon2 gAactccaaagattcaggtttactcacgtcatccagcagagaatggaaagtcaaatttcctgaattgctatgtgtctgggtttcatccatccgacattgaagttgacttactgaagaatggagagagaattgaaaaagtggagcattcagacttgtctttcagcaaggactggtctttctatctcttgtactacactgaattcacccccactgaaaaagatgagtatgcctgccgtgtgaaccatgtgactttgtcacagcccaagatagttaagtggg 5 hB2M - intron2 gtaagtcttacattcttttgtaagctgctgaaagttgtgtatgagtagtcatatcataaagctgctttgatataaaaaaggtctatggccatactaccctgaatgagtcccatcccatctgatataaacaatctgcatattgggattgtcagggaatgttcttaaagatcagattagtggcacctgctgagatactgatgcacagcatggtttctgaaccagtagtttccctgcagttgagcagggagcagcagcagcacttgcacaaatacatatacactcttaacacttcttacctactggcttcctctagcttttgtggcagcttcaggtatatttagcactgaacgaacatctcaagaaggtataggcctttgtttgtaagtcctgctgtcctagcatcctataatcctggacttctccagtactttctggctggattggtatctgaggctagtaggaagggcttgttcctgctgggtagctctaaacaatgtattcatgggtaggaacagcagcctattctgccagccttatttctaaccattttagacatttgttagtacatggtattttaaaagtaaaacttaatgtcttccttttttttctccactgtctttttcatag 6 hB2M - exon3 atcgagacatg 7 <![CDATA[linker (G4S)3]]> GGTGGCGGAGGGAGTGGAGGGGGTGGCAGCGGAGGCGGTGGGAGT 8 HLA-A*0201-α1 ggctctcactccatgaggtatttcttcacatccgtgtcccggcccggccgcggggagccccgcttcatcgcagtgggctacgtggacgacacgcagttcgtgcggttcgacagcgacgccgcgagccagaggatggagccgcgggcgccgtggatagagcaggagggtccggagtattgggacggggagacacggaaagtgaaggcccactcacagactcaccgagtggacctggggaccctgcgcggctactacaaccagagcgaggcc 9 HLA-A*0201-α2 ggttctcacaccgtccagaggatgtatggctgcgacgtggggtcggactggcgcttcctccgcgggtaccaccagtacgcctacgacggcaaggattacatcgccctgaaagaggacctgcgctcttggaccgcggcggacatggcagctcagaccaccaagcacaagtgggaggcggcccatgtggcggagcagttgagagcctacctggagggcacgtgcgtggagtggctccgcagatacctggagaacgggaaggagacgctgcagcgcacgg 10 mouse HLA-A*0201-α3 + transmembrane region + intracellular region attccccaaaggcacatgtgacccatcaccccagatctaaaggtgaagtcaccctgaggtgctggggccctgggcttctaccctgctgacatcaccctgacctggcagttgaatggggaggagctgacccaggacatggagcttgtggagaccaggcctgcaggggatggaaccttccagaagtgggcatctgtggtggtgcctcttgggaaggagcagaattacacatgccgtgt gtaccatgaggggctgcctgagcccctcaccctgagatgggagcctcctccgtccactgactcttacatggtgatcgttgctgttctgggtgtccttggagctatggccatcattggagctgtggtggcttttgtgatgaagagaggagaaacacaggtggaaaggagggactatgctctggctccaggctcccagagctctgaaatgtctctccgagattgtaaagcgtga 11 WPRE polyA ccgtatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgataccgtcgatcctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggga 12 loxp-PGK-puro-polyA-loxp GAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCATCGATaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgctacacaagtggcctctggcctcgcacacattccacatcccccggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggtccgggggcgggctcaggggcgggctcaggggcggggcgggcgcccgaaggtcctccggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcagcccaagctagcttaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgaggtacctctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcacCgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatCGATGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC 13 Example 3: Obtaining humanized HLA mouse embryonic stem cells

[0072] 3.1 Stem cell targeting C57BL6 / J mouse embryonic stem cells were revived from a liquid nitrogen frozen cell bank, specifically using mouse embryonic stem cells within passage number p10, and cultured in 6 cm culture dishes for 3 days in 15% serum + LIF + 2i: Knockout DMEM + 15% FBS + NEAA + Gluamax + β-me + LIF + pD0325901 + chir99021 + Feeder plating conditions. Approximately 2 × 10 cells were electroporated using the Nucleofector™ IIs / 2b electroporator and the Mouse ES Cell Nucleofector® Kit (Lonza, VPH-1001) using the A023 program. 6The cells were electroporated, and the electroporation process was carried out in 100 μL of electroporation buffer containing the linearized targeting vector prepared in Example 2, the Cas12 protein, and the crRNA1 and crRNA2 prepared in Example 1. The transfected cells were seeded into three wells of a 6-well plate and then recovered for 36 hours. After recovery, 1 μg / mL puromycin (Merck) was added to the cell culture medium. After 3 days of screening, puromycin-resistant mouse embryonic stem cell clones were picked and cultured in 96-well plates by using a glass needle to draw single clones. The cultured cell clones were subjected to PCR to screen for positive clones.

[0073] PGK-Puro selection marker deletion: Mouse embryonic stem cells with positive PCR results were cultured in 6 cm dishes and electroporated using the Nucleofector™ IIs / 2b electroporator (using the A023 program) and the Mouse ES CellNucleofector® Kit (Lonza, VPH-1001) to produce approximately 2 × 10 6 Cells were electroporated in 100 μL of electroporation buffer containing the pPGK-FLPo plasmid (Addgene, 13793). The transfected cells were seeded into six wells of a 12-well plate. Three days later, single clones were extracted using a glass needle and cultured in 96-well plates. The next day, the clones were digested and subcultured, split in half: one undergoing puromycin selection and the other undergoing normal culture. If the PGK-Puro resistance selection marker is successfully deleted, cells will die after puromycin selection due to intolerance. The clones corresponding to puromycin intolerance are the successfully edited humanized HLA mouse embryonic stem cells.

[0074] 3.2 Genotyping of Humanized HLA Mouse Embryonic Stem Cells Successfully edited single clones obtained in step 3.1 were trypsinized and cultured in duplicate. One aliquot of cells was lysed in 10 μL of NP 40 lysis buffer (NP40 lysis buffer: 10 mL TE (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA) + 0.5% NP40 + 10 μL 10 mg / mL proteinase K) at 56°C for 60 minutes, followed by lysis at 95°C for 10 minutes. The lysate was used as a template for genotyping PCR screening. PCR was performed using Phanta Max Super-Fidelity DNA Polymerase (Novozymes) according to the manufacturer's instructions.

[0075] PCR analysis was performed for homologous recombination (HDR) targeting the mouse HLA locus with 5' and 3' homology arms. The genotype identification scheme is shown in Table 2 below. The identification of the targeted insertion primers and the identification results are shown in Table 2. Figures 3 - 4 As shown. If the recombinant vector is inserted into the correct position, a band identical to the expected one should appear. According to the PCR gel image of the corresponding primers, it can be judged that if 5'-KI is successful, a band of 1888bp will appear, and if 3'-KI is successful, a band of 2016bp (puro) or 2090bp (BSD) will appear; if it is a homozygous KI, a 1661bp WT band and a 723bp KO band should not appear. According to Figures 3 - 4 Homozygous KI embryonic stem cell lines that meet the expected results, such as A5, E5, F5, G5, G6, and H6, are selected for further resistance deletion. The FRT-PGK-puro-polyA-FRT or FRT-PGK-BSD-polyA-FRT resistance expression cassette contained in the KI sequence is deleted using FLP recombinase, ultimately leaving only an FRT site.

[0076] Table 2 ; The PCR products of the positive cell lines with the correct insertion position of the recombinant vector and deletion resistance were sent for testing. The PCR scheme and editing site detection scheme are shown in Table 3 below. Figure 5 shown.

[0077] Table 3 ; The full length of the sequence tested is shown in SEQ ID NO: 30 below, which is the sequence of the successful insertion (chimeric gene): The nucleotide sequence of SEQ ID NO: 30 encodes the following amino acid sequence shown in SEQ ID NO: 31: TPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGS HSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDSPKAHVTHHPRSKGEVTLRCWALGFYPADITLTWQLNGEELTQDM ELVETRPAGDGTFQKWASVVVPLGKEQNYTCRVYHEGLPELPLTLRWEPPPSTDSYMVIVAVLGVLGAMAIIGAVVAFVMKRRRNTGGKGGDYALAPGSQSSEMSLRDCKA (SEQ ID NO: 31).

[0078] Example 4: Obtaining humanized HLA mice through tetraploid embryo compensation technology 4.1 Tetraploid Compensation Technology Embryo transfer was performed through a tetraploid compensation experiment. The specific steps are as follows: 1) Intraperitoneally inject 7.5 units of pregnant mare serum gonadotropin (PMSG) into 4-10 week old B6C3F1 female mice. 48 hours later, inject human chorionic gonadotropin (hCG) and house them with CD1 male mice. The next morning, examine the females for vaginal plugs and remove any with plugs. Record the corresponding fertilization time.

[0079] 2) The next day, euthanize the pregnant mouse by cervical dislocation. Disinfect the abdomen with 70% alcohol. Use auxiliary forceps and ophthalmic scissors to cut through the abdominal skin and muscle layer, opening the abdominal cavity. Grasp the upper portion of one uterine horn with forceps and use scissors to make a small incision in the membrane adjacent to the oviduct. Cut the oviduct and ovary at their junction. Transfer the oviduct and attached uterus to a 35 mm culture dish. Secure the oviduct fimbria with forceps. Gently insert an irrigation needle filled with M2 medium into the fimbria. Irrigate the oviduct with 0.1 mL of M2 medium. Collect the embryos flushed out with an oviduct transfer tube and rinse three times with M2 medium. Collect E1.5 mouse 2-cell embryos. For detailed experimental procedures, refer to Hogan, B. (1994). Manipulating the mouse embryo: a laboratory manual, 2nd edn (Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press).

[0080] 3) The collected mouse embryos were placed in 0.3 M mannitol containing 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.3% bovine serum albumin. Electrofusion was performed using a Cellfusion CF-150 / B electrofusion apparatus and a 250-μm fusion tank (BLS Ltd., Budapest, Hungary) at 60 V for 50 μs to obtain quadruple embryos. The embryos were then placed in KSOM medium (Summers, MC, McGinnis, LK, Lawitts, JA, Raffin, M., and Biggers, JD (2000). IVF of mouse ova in a simple x optimized medium supplemented with amino acids. HumReprod 15, 1791-1801.), cultured in a CO2 incubator for 24 hours, and then the zona pellucida was removed with acidic Tyrode's solution (Sigma-Aldrich, T1788). The cells were aggregated with embryonic stem cells (i.e., humanized HLA mouse embryonic stem cells prepared in Example 3) to form chimeric embryos (Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, JC (1993). Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 90, 8424-8428.).

[0081] 4) The chimeric embryos were cultured overnight in a CO2 incubator and transplanted into the uterus of a pseudo-pregnant mouse on day E2.5. After 17 days, the surrogate mouse was euthanized by cervical dislocation and a laparotomy was performed. The living and breathing newborn mice were placed in a nurse mouse cage and weaned after 21 days to obtain transgenic mice completely derived from embryonic stem cells, namely humanized HLA gene-modified mice (HLA-A*0201 humanized mice).

[0082] 4.2 Mouse genotype identification: After the mouse tail was lysed, PCR was performed as a template. The identification scheme is shown in Table 4. The identification results of different mice are shown in Figure 6 As shown, it shows that humanized HLA gene-modified mice (HLA-A*0201 humanized mice) were successfully constructed.

[0083] Table 4

[0084] Example 5: FACS detection of HLA-A*0201 humanized mouse related gene expression 5.1 Flow cytometry experimental steps: Peripheral blood was obtained from male C57BL / 6 and humanized mice (n=3, 8 weeks old) and analyzed by flow cytometry to assess leukocyte subsets.

[0085] The specific steps are as follows: 1) Obtain a single-cell suspension: Place 200 μL of peripheral blood sample in a 15 mL centrifuge tube and add 5 mL of ACK-gibco (erythrocyte lysis buffer). Lyse at room temperature for 10 min. Add 2 volumes of PBS, mix well, and centrifuge at 10°C, 350 g, and 5 min. 2) Cell counting: Discard the supernatant and resuspend in 100-200 μL FACS Buffer (2% FBS); count cells between each reaction; take 5×10 5 For about 100 cells, add FACS Buffer to 100 μL / reaction; 3) Fc blocking: Add 3-5 μL of Fc blocking solution (Anti-Mo CD16 / CD32) according to the cell volume and block on ice for 15 minutes. The general volume ratio of Fc is 1:100, and the stock solution concentration is 0.5 mg / mL. 4) Antibody Staining: All antibodies used in the array (all purchased from Biolegend, including: mouse B2M antibody, Catalog No. 154503; H-2Db antibody, Catalog No. 111507; human B2M antibody, Catalog No. 395711; HLA-A2.1 antibody, Catalog No. 343307; mouse CD8 antibody, Catalog No. 100707; mouse CD4 antibody, Catalog No. 100411; mouse CD45 antibody, Catalog No. 157213) were added to 1.5 mL EP tubes according to the color combination, mixed thoroughly, and placed on ice. Human antibodies are usually 5 μL / Test, and mouse antibodies are usually 1 μL / Test. Stain on ice in the dark for 15 min, gently tapping the bottom of the tube 1-2 times to mix thoroughly. 5) Stop staining: Add 1 mL of FACS Buffer to wash away unbound antibody-conjugated dye, invert to mix, and centrifuge at 350 g for 5 min at 10°C. Aspirate the supernatant, leaving approximately 50-100 μL. 6) DAPI staining: Add 400 μL of DAPI staining solution, resuspend and mix thoroughly, stain for 1 minute, then add 1 mL of FACS buffer and mix thoroughly. Centrifuge at 350 g for 5 minutes at 10°C to remove unbound DAPI. Aspirate the supernatant, leaving approximately 50-100 μL. 7) Resuspend and analyze: Use a BD LSR Fortessa SORP analytical flow cytometer. Resuspend the sample in 400 μL FACS Buffer (DPBS + 2% FBS). Filter through a 300-mesh sieve into a flow cytometer tube. Place on ice and protect from light before analysis.

[0086] 5.2 Analysis of protein expression in blood like Figure 7 As shown, mouse mB2M and H2D were detected in the blood of wild-type C57BL / 6J mice (+ / +) and HLA-A*0201 humanized mice (H / H). b Human hB2M and HLA-A2 were only detected in HLA-A*0201 humanized mice, while mouse mB2M and H-2D were not detected. b This indicates that the mB2M of the HLA-A*0201 humanized mouse was successfully deleted, HLA-A2 and hB2M were successfully expressed, and H2D b Not expressed.

[0087] 5.3 Analysis of T cell ratio in blood: like Figure 8 As shown, the percentages of CD8+ T cells in the blood of wild-type C57BL / 6J mice (+ / +) and HLA-A*0201 humanized mice (H / H) are basically the same, indicating that the optimized HHD regimen used in the HLA-A*0201 homozygous humanized mice obtained in the present disclosure has little effect on the development of CD8+ T cells and can successfully activate the endogenous CD8+ T cells of mice.

[0088] The flow cytometry data of this example showed that HLA-A2 and hB2M were successfully expressed in HLA-A*0201 humanized mice; mB2M was successfully deleted, and H2D b No expression; the mouse's CD8+ T cells can be activated. Therefore, HLA-A*0201 humanized mice were successfully generated and showed high CD8+ T cell activation levels, which has important application value for mouse breeding as a product.

[0089] Example 6 Obtaining HLA-A*2402 Humanized Mice Other different HLA typing also used the same humanized gene editing scheme as Examples 2-4. Compared with the aforementioned HLA-A*0201 humanized mouse, only the human α1 and α2 sequences of HLA-A, and the mouse α3+transmembrane region+intracellular region sequences were different, that is, they were replaced with sequences expressing the typing.

[0090] In this example, humanized HLA mice with HLA-A*2402 typing (HLA-A*2402 humanized mice) were prepared, wherein HLA-A*2402-α1, HLA-A*2402-α2, HLA-A*2402-α3 intron, and HLA-A*2402-α3+transmembrane region+intracellular region are shown in Table 5 below.

[0091] Table 5 ; The prepared HLA-A*2402 humanized mice were genotyped and the identification primers were shown in Table 2. The mouse gene sequencing results were shown in Figure 9 shown.

[0092]

[0093] It encodes the following amino acid sequence shown in SEQ ID NO: 37: TPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSG SHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYH QYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDSPKAHVTHHPRSKGEVTLRCWALGFYPADITLTWQLNGEELTQ DMELVETRPAGDGTFQKWASVVVPLGKEQNYTCRVYHEGLPELPLTLRWEPPPSTDSYMVIVAVLGVLGAMAIIGAVVAFVMKRRRNTGGKGGDYALAPGSQSSEMSLRDCKA.

[0094] Example 7 FACS detection of HLA-A*2402 humanized mouse related gene expression The expression of HLA-A*2402 humanized mouse related genes was detected by the same method as in Example 5. The flow cytometry verification results were as follows: Figure 10 The results showed that mouse mB2M and H2D could be detected in the blood of wild-type C57BL / 6J mice (+ / +) and HLA-A*2402 humanized mice (H / H). b Human hB2M was only detected in HLA-A*2402 humanized mice, while mouse mB2m and H-2D were not detected. b This indicates that the mB2m of HLA-A*2402 humanized mice was successfully deleted, hB2M was successfully expressed, and H2D b In addition, the percentages of CD8+ T cells in the blood of wild-type C57BL / 6J mice (+ / +) and HLA-A*2402 humanized mice (H / H) were similar, indicating that the optimized HHD regimen used in the HLA-A*2402 homozygous humanized mice obtained in this disclosure has little effect on the development of CD8+ T cells and can successfully activate endogenous CD8+ T cells in mice.

[0095] The flow cytometry data of this example showed that hB2M was successfully expressed in HLA-A*2402 humanized mice; mB2m was successfully deleted, and H2D b No expression; the mouse's CD8+ T cells can be activated. Therefore, HLA-A*2402 humanized mice were successfully generated and showed high CD8+ T cell activation levels, which has important application value for mouse breeding as a product.

[0096] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for establishing transgenic mice, characterized in that: The method comprises: introducing a transgenic mouse MHC chimeric gene into the mouse endogenous B2m gene locus, wherein the MHC chimeric gene comprises a human B2M gene and a chimeric α chain gene; The chimeric α chain gene includes a human α1 region, a human α2 region, and a mouse α3 region; The chimeric α chain gene further includes a mouse transmembrane region and a mouse intracellular region.

2. The method according to claim 1, characterized in that The cells of the transgenic mouse express human B2M protein and chimeric α chain; and / or The expression of endogenous B2m protein in the cells of the transgenic mice is reduced or absent.

3. The method according to claim 1, characterized in that The MHC chimeric gene replaces the nucleotide sequence of exon 2 to exon 3 of the mouse endogenous B2m gene.

4. The method according to claim 1, wherein The chimeric α chain gene comprises nucleotide sequences encoding human HLA α1 and α2; and / or The chimeric α chain gene further comprises nucleotide sequences encoding α3, transmembrane region and intracellular region of mouse H2D protein; and / or The human B2M gene comprises the nucleotide sequences of exon 2, intron 2 and exon 3 of human B2M; and / or The human B2M gene and the chimeric α chain gene are connected via a linker sequence encoding a linking function.

5. The method according to claim 4, characterized in that The human HLA is selected from human HLA-A, human HLA-B or human HLA-C; the human HLA-A includes HLA-A*02:01, HLA-A*24:02, HLA-A*01:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:04, HLA-A*03:01, HLA-A*11:

01. HLA-A*23:01, HLA-A*25:01, HLA-A*26:01, HLA-A*29:02, HLA-A*30:01, HLA-A* 31:01, HLA-A*32:01, HLA-A*33:01, HLA-A*68:01, HLA-A*68:02, or HLA-A*69:01; and / or The MHC chimeric gene comprises the nucleotide sequence as shown in SEQ ID NO: 30 or 36, or a nucleotide sequence having at least 90% sequence identity thereto, or a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides thereto; and / or The transgenic mouse has the amino acid sequence shown in SEQ ID NO: 31 or 37; and / or The transgenic mice are transgenic mice.

6. The method according to claim 1, characterized in that The method comprises: using a recombinant vector of the B2M gene to replace exon 2 and exon 3 of the mouse endogenous B2m gene; The recombinant vector of the B2m gene comprises an MHC chimeric gene, a 3' homology arm and a 5' homology arm; and / or The replacement also uses the crRNA / cas12 system, wherein the targeting site of the crRNA is located on intron 1 of the mouse B2m gene and / or on the intron between exon 3crRNA / cas12 system-4; and / or the sequence of the crRNA includes the sequence shown in SEQ ID NO: 1 and / or the sequence shown in SEQ ID NO:

2.

7. The method according to claim 1, characterized in that The method comprises the following steps: (1) Determine the target site based on the sequence of intron 1 and the intron between exons 3-4 of the mouse B2m gene; (2) Synthesizing the crRNA sequence according to the target site determined in step (1); (3) constructing a linear targeting vector containing the MHC chimeric gene; (4) introducing the crRNA obtained in step (2) and the linear targeting vector obtained in step (3) into mouse embryonic stem cells to obtain mouse embryonic stem cells that express human B2M protein and MHC chimeric α chain and have reduced or absent endogenous B2m gene expression; (5) The mouse embryonic stem cells obtained in step (4) are aggregated with tetraploid cells to prepare transgenic mice.

8. The method according to claim 7, characterized in that In step (2), the crRNA comprises the sequence shown in SEQ ID NO: 1 and / or the sequence shown in SEQ ID NO: 2; and / or In step (5), the transgenic mice are prepared by tetraploid compensation technology.

9. A transgenic mouse tissue, body fluid, cell, or fragment thereof, or an extract thereof, characterized in that: The transgenic mouse is constructed using the method according to any one of claims 1 to 8 or is a progeny thereof, the cells or tissues cannot develop into individual animals, and the cells do not include germ cells.

10. The transgenic mouse tissue, body fluid, cell, or fragment thereof or extract thereof according to claim 9, characterized in that: The genome of the cells or tissues of the transgenic mouse comprises the nucleotide sequence as shown in SEQ ID NO: 30 or 36, or a nucleotide sequence having at least 90% sequence identity thereto, or a nucleotide sequence comprising a substitution, deletion and / or insertion of one or more nucleotides compared thereto; and / or The cells or tissues of the transgenic mouse include the amino acid sequence shown in SEQ ID NO: 31 or 37; and / or The CD8+ T cell content of the transgenic mice is comparable to that of wild-type mice.

11. A chimeric gene, characterized in that The chimeric gene comprises a human B2M gene and a chimeric α chain gene.

12. The chimeric gene according to claim 11, characterized in that The chimeric gene comprises the nucleotide sequence shown in SEQ ID NO: 30 or 36, or a nucleotide sequence having at least 90% sequence identity thereto, or a nucleotide sequence comprising substitution, deletion and / or insertion of one or more nucleotides compared thereto.

13. A recombinant vector, characterized in that The recombinant vector comprises the chimeric gene according to claim 11 or 12.

14. The recombinant vector according to claim 13, characterized in that The recombinant vector further comprises a 3' homology arm and a 5' homology arm, wherein the 3' homology arm is shown in SEQ ID NO: 3, and the 5' homology arm is shown in SEQ ID NO:

4.

15. The method according to any one of claims 1 to 8, the chimeric gene according to claim 11 or 12, or the recombinant vector according to claim 13 or 14, Applications in product development requiring immune processes involving human cells, manufacturing human antibodies, vaccines, or as model systems for pharmacology, immunology, microbiology, and medical research; Application in the production and use of animal experimental disease models for human cell transplantation, immune system reconstruction, and pathogenicity research; Application in the formation and functional study of human hematopoietic stem cells and / or in the construction of disease models; or Application in screening, verification, evaluation or research of MHC function, human MHC signaling mechanism, human-targeted antibodies, vaccines, human-targeted drugs, drug efficacy, immune-related disease drugs, and anti-tumor or anti-inflammatory drugs, screening and evaluation of human drugs and drug efficacy research.

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