Humanized animals of MHC gene group

Abstract

The present invention relates to a carrier group comprising a combination of the following carriers (A), (B) and (D) or (C) and (D): (A) a targeting carrier 1 introduced to an arbitrary position P1 on the distal side of the human MHC region; (B) a targeting carrier (2) introduced to a position (p1) corresponding to the P1 in a non-human MHC region; (C) a targeting vector 3 that causes recombination between a region from the P1 to the distal end and a region from the p1 to the distal end; and (D) a targeting vector (4) that causes recombination in the recombined chromosome between a region from an arbitrary position (P2) on the proximal side of the human MHC region to the distal end and a region from a position (p2) corresponding to the P2 in a chromosome in which non-human MHC is present to the distal end.

Description

Technical Field

[0001] The present invention relates to a non-human mammal whose major histocompatibility gene complex (MHC) has been humanized and a method for producing the same. Background Art

[0002] Acquired immunity is an immune system that activates T cells and B cells and eliminates pathogens, virus-infected cells, cancer cells, etc. by the coordinated action of three factors: MHC, T cell receptors, and antibodies. Acquired immunity can target specific molecules and has been used in the development of biopharmaceuticals. Antibody medicines are the representative of these, and can be used to treat various diseases such as cancer. It is said that the market size will be close to 18 trillion yen by 2020 (Non-patent Documents 1, 2).

[0003] However, antibodies can only recognize extracellular antigens, so some people point out that promising target molecules are being depleted. Therefore, the current focus is on the development of pharmaceuticals using T cell receptors (TCRs) (non-patent literature 3). If it is TCR, intracellular proteins can also become targets, so a lot of molecules can become targets. Clinical trials have been conducted on pharmaceuticals that have used more TCRs, but there are also problems in drug development using TCRs, one of which is that there is no appropriate screening system using organisms.

[0004] The three factors of MHC, TCR and antibody that play a major role in acquired immunity have diversity between species, so it is difficult to produce medicines utilizing acquired immunity using model animals. Therefore, in recent years, preparation has been made to humanize these genes in mice and used for drug development, etc. (non-patent literature 4-8), and only the humanization of MHC gene group has not been realized. In the case where MHC is derived from mice, even if only TCR is humanized, mouse MHC restricted TCR will be produced, so it is a more difficult situation to use mice to screen sufficient TCR.

[0005] The reason why humanization of only the MHC gene group has not been achieved is that humanization is very difficult technically. There are 21 MHC genes in humans and 39 in mice, and these genes are mixed with various genes in a genomic region of about 3 to 4 million base pairs called the MHC region.

[0006] Bacterial artificial chromosomes (BAC) can be used as vectors that can retain more sequences and are used for the interpretation of genome sequences, etc. However, even if BAC is used, it can only retain about 200,000 base pairs, and it is very difficult to introduce the MHC region as a whole through conventional transgenic technology. A method of replacing the human MHC region with cDNA of the human MHC gene group is also considered. In this case, only transgenic mice of various human MHC genes can be prepared, and mice expressing all human MHC genes can be prepared through mating. It takes at least about 6 months to establish a transgenic mouse system and make it a state that can mate, so it is actually very difficult to use conventional genetic engineering technology to prepare MHC gene group humanized mice.

[0007] Therefore, when mice expressing HLA are required, transgenic mice into which only specific human MHC has been introduced can be used (Non-patent Documents 9, 10). However, since MHC genes are subject to expression control depending on the environment, the introduction of cDNA that does not contain the expression control region around the gene locus cannot accurately reproduce the human immune response.

[0008] Prior art literature

[0009] Non-patent literature

[0010] Non-patent literature 1: Goydel, RS and Rader, C. (2021) Antibody-based cancer therapy. Oncogene, 40, 3655-3664.

[0011] Non-patent literature 2: Zahavi, D. and Weiner, L. (2020) Monoclonal Antibodies in Cancer Therapy. Antibodies (Basel), 9.

[0012] Non-patent literature 3: Zhao, L. and Cao, YJ (2019) Engineered T Cell Therapy for Cancer in the Clinic. Front Immunol, 10, 2250.

[0013] 4:Tomizuka,K.,Shinohara,T.,Yoshida,H.,Uejima,H.,Ohguma,A.,Tanaka,S.,Sato,K.,Oshimura,M.and Ishida,I.(2000)Doubletrans-chromosomic mice:maintenance of two individual human chromosome e fragments containing Ig heavy and kappa loci and expression of fully human antibodies.Proc Natl Acad Sci US A,97,722-727.

[0014] Polymer 5:Tomizuka,K.,Yoshida,H.,Uejima,H.,Kugoh,H.,Sato,K.,Ohguma,A.,Hayasaka,M.,Hanaoka,K.,Oshimura,M.and Ishida,I.(1997)Functional expressionand germline transmission of a human chromosome fragment in chimaericmics.Nat Genet,16,133-143.

[0015] Materials 6: Li, LP, Lampert, JC, Chen, X., Leitao, C., Popovic, J., Muller, W. and Blankenstein, T. (2010)Transgenic mice with a diverse human T cellantigen receptor repertoire.Nat Med,16,1029-1034.

[0016] Obenaus, M., Leitao, C., Leisegang, M., Chen, X., Gavv ovidis, I., Van der Bruggen, P., Uckert, W., Schendel, DJand Blankenstein, T. (2015). affinity to cancer antigens using antigen-negative humanized mice.Nat Biotechnol,33,402-407.

[0017] SPECIFICATIONS 8:Poncette,L.,Chen,X.,Lorenz,FKand Blankenstein,T.(2019)Effective NY-ESO-1-specificMHC II-restricted T cell receptors from antigen-negative hosts enhance tumor regression.J Clin Invest,129,324-335.

[0018] Materials 9:Shultz, LD, Saito, Y., Najima, Y., Tanaka, S., Ochi, T., Tomizawa, M., Doi, T., Sone, A., Suzuki, N., Fujiwara, H. et al HLA-restricted immune responses in HLA-expressing NOD / SCID / IL2r gamma(null)humanized mice.Proc Natl Acad Sci U SA,107,13022-13027.

[0019] Non-patent literature 10: Suzuki, M., Takahashi, T., Katano, I., Ito, R., Ito, M., Harigae, H., Ishii, N. and Sugamura, K. (2012) Induction of human humoral immune responses in a novel HLA-DR-expressing transgenic NOD / Shi-sci d / γcnull mouse.IntImmunol,24,243-252. Summary of the invention

[0020] Technical problem solved by the invention

[0021] When comparing the MHC region of humans and mice, it is known that the MHC region is huge, and if species-specific genes are removed, the gene composition in the region is maintained in both. Therefore, it is believed that if the MHC region as a whole is replaced with humans and mice, MHC can be fully humanized. However, it is impossible to use a universal gene vector system to introduce such a huge sequence, and the previous technology cannot be used to replace the MHC region. Thus, it is expected to develop a targeting vector for replacing the MHC of non-human mammals with the MHC of people and a technology for replacing the MHC of non-human mammals with the MHC of people by utilizing the targeting vector.

[0022] Technical means of solving problems

[0023] The present inventors have conducted intensive studies to solve the above problems and, as a result, have succeeded in replacing the MHC of a non-human mammal with the MHC of a human, thereby completing the present invention.

[0024] That is, the present invention is as follows:

[0025] [1] A vector set comprising a combination of the following targeting vectors (A), (B) and (D) or a combination of the following targeting vectors (C) and (D):

[0026] (A) A targeting vector 1 is introduced into an arbitrary position P1 on the distal side of the major histocompatibility complex (human MHC) region present on the short arm of human chromosome 6,

[0027] The targeting vector 1 causes translocation recombination between the region from the P1 to the distal end and the region from the position p1 corresponding to the P1 to the distal end, and the targeting vector 1 comprises a gene cassette 1, the position p1 is present on the distal side of the MHC (non-human MHC) region of the chromosome in a non-human mammal, and the gene cassette 1 comprises a recombinase recognition sequence, a partial sequence of a drug resistance gene 1 for drug screening reconstructed during translocation recombination, and a drug resistance gene 2 for drug screening after being targeted;

[0028] (B) a targeting vector 2, which is introduced into a chromosome of a non-human mammal having an MHC region, into a position p1 corresponding to the P1 on the distal side of the MHC (non-human MHC) region,

[0029] The targeting vector 2 causes translocation recombination between the region from the p1 to the distal end and the region from the P1 to the distal end in human chromosome 6, and the targeting vector 2 comprises a gene cassette 2, wherein the gene cassette 2 comprises a recombinase recognition sequence, other sequences of the drug resistance gene 1, and a drug resistance gene 3 for drug screening after targeting;

[0030] (C) a targeting vector 3 that causes translocation recombination between a region from an arbitrary position P1 to the distal end of the distal side of the human MHC region in human chromosome 6, which is a target for cutting by a genome editing tool, and a region from a position p1 to the distal end of the distal side of the non-human MHC region in a chromosome of a non-human mammal having MHC, which corresponds to the position P1,

[0031] The targeting vector 3 comprises a sequence identical to the sequence of a portion of the region R1a between the P1 and the human MHC region, a drug resistance gene 4 for drug screening after being targeted, and a sequence identical to the sequence of a portion of the region R2b on the distal side of the P1, or comprises a sequence identical to the sequence of a portion of the region R1b on the distal side of the P1, a drug resistance gene 4 for drug screening after being targeted, and a sequence identical to the sequence of a portion of the region R2a between the P1 and the non-human MHC region;

[0032] (D) a targeting vector 4 that causes translocation recombination between a region from an arbitrary position P2 on the proximal side of the human MHC region that is a target for cleavage by a genome editing tool to the distal end and a region from a position p2 on the proximal side of the non-human MHC region corresponding to P2 to the distal end in a chromosome of a non-human mammal having non-human MHC,

[0033] The targeting vector 4 contains the same sequence as the sequence of a portion of the region R3a on the proximal side of the P2, a drug resistance gene 5 for drug screening after targeting, and the same sequence as the sequence of a portion of the region R4b between the P2 and the non-human MHC region, or contains the same sequence as the sequence of a portion of the region R3b between the P2 and the human MHC region, a drug resistance gene 5 for drug screening after targeting, and the same sequence as the sequence of a portion of the region R4a on the proximal side of the P2.

[0034] [2] The vector set according to [1], further comprising the following vectors (E) and (F):

[0035] (E) a genome editing tool vector 1, which cuts human chromosome 6 at the arbitrary position P1;

[0036] (F) A genome editing tool vector 2, which cuts the non-human chromosome at the arbitrary position p1.

[0037] [3] The vector set according to [1], further comprising the following vectors (G) and (H):

[0038] (G) a genome editing tool vector 3, which cuts human chromosome 6 at the arbitrary position P2;

[0039] (H) A genome editing tool vector 4, which cuts the non-human chromosome at the arbitrary position p2.

[0040] [4] The vector set according to any one of [1] to [3], wherein

[0041] The human MHC region includes a class I region, a class II region, a class III region, or a combination thereof.

[0042] [5] The vector set according to [4], wherein

[0043] The class I region comprises at least one selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, MICA, and MICB.

[0044] [6] The vector set according to [4], wherein

[0045] The class II region includes at least one selected from HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO.

[0046] [7] The vector set according to [4], wherein

[0047] A region containing a class I region is a region containing HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, MICA, and MICB.

[0048] [8] The vector set according to [4], wherein

[0049] The region containing the class II region is a region containing HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO.

[0050] [9] The vector set according to [4], wherein

[0051] The region including both the class I region and the class II region is the region between the DAXX locus and the MOG locus.

[0052]

[10] The vector set according to any one of [1] to [3], wherein

[0053] The arbitrary position P1 is the position between the MOG locus and the GABBR1 locus.

[0054]

[11] The vector set according to any one of [1] to [3], wherein

[0055] The arbitrary position P2 is the position between the KIFC1 locus and the DAXX locus.

[0056]

[12] The vector set according to any one of [1] to [3], wherein

[0057] Non-human mammals are rodents.

[0058]

[13] The vector set according to

[12] , wherein

[0059] The rodent is a mouse.

[0060]

[14] The vector set according to

[13] , wherein

[0061] The arbitrary position p1 is the position between the Mog locus and the Gabbr1 locus.

[0062]

[15] The vector set according to

[13] , wherein

[0063] The arbitrary position p2 is the position between the Kifc1 locus and the Daxx locus.

[0064]

[16] The vector set according to any one of [1] to [3], wherein

[0065] Drug resistance gene 1 is a neomycin resistance gene, drug resistance gene 2 is a hygromycin resistance gene and / or drug resistance gene 3 is a puromycin resistance gene.

[0066]

[17] The vector set according to any one of [1] to [3], wherein

[0067] The drug resistance gene 4 is a neomycin resistance gene and / or the drug resistance gene 5 is a blasticidin resistance gene.

[0068]

[18] The vector set according to any one of [1] to [3], wherein

[0069] The recombinase recognition sequence in gene cassette 1 is loxP, drug resistance gene 1 is a neomycin resistance gene, and drug resistance gene 2 is a hygromycin resistance gene.

[0070]

[19] The vector set according to any one of [1] to [3], wherein

[0071] Gene cassette 1 comprises a construct represented by the following formula (1):

[0072] BGHpA-3'Neo-SA-loxP-EF1-Hyg-P2A-mRuby2-RGpA(1)

[0073] In the formula, BGHpA represents the poly A tailing signal sequence derived from bovine growth hormone, 3'Neo represents the sequence of a part of the 3' end of the neomycin resistance gene, SA represents the splice acceptor sequence, loxP represents the recombinase recognition sequence, EF1 represents the promoter sequence derived from human elongation factor 1α, Hyg represents the hygromycin resistance gene, P2A represents the 2A sequence derived from porcine teschovirus, mRuby2 represents the red fluorescent gene, and RGp A represents the poly A tailing signal sequence derived from rabbit β-globin.

[0074]

[20] The vector set according to any one of [1] to [3], wherein

[0075] The recombinase recognition sequence in gene cassette 2 is loxP, drug resistance gene 1 is a neomycin resistance gene, and drug resistance gene 3 is a puromycin resistance gene.

[0076]

[21] The vector set according to any one of [1] to [3], wherein

[0077] Gene cassette 2 comprises a construct represented by the following formula (2):

[0078] EF1-EGFP-P2A-5'Neo-SD-loxP-SA-T2A-Puro-RGpA(2)

[0079] In the formula, EF1 represents the promoter sequence derived from human elongation factor 1α, EGFP represents the green fluorescent gene, P2A represents the 2A sequence derived from porcine teschovirus, 5'Neo represents the sequence of a part of the 5' end of the neomycin resistance gene, SD represents the splice donor sequence, loxP represents the recombinase recognition sequence, SA represents the splice acceptor sequence, T2A represents the 2A sequence derived from Thosea asigna virus, Puro represents the puromycin resistance gene, and RGpA represents the poly A tailing signal sequence derived from rabbit β-globin.

[0080]

[22] A non-human mammalian cell in which the MHC region has been humanized, comprising:

[0081] A chromosome obtained by replacing the MHC region of a non-human mammal chromosome with the MHC region of a human chromosome using the vector set described in any one of [1] to

[21] .

[0082]

[23] The cell according to

[22] , which is an ES cell.

[0083]

[24] A non-human mammal with a humanized MHC region, produced by the cell of

[22] or

[23] .

[0084]

[25] The non-human mammal according to

[24] , wherein

[0085] Non-human mammals are rodents.

[0086]

[26] The non-human mammal according to

[25] , wherein

[0087] The rodent is a mouse.

[0088]

[27] A non-human mammal according to

[26] , comprising: a chromosome obtained by replacing the chromosome portion of at least the portion of ...

[0089]

[28] A method for producing a non-human mammal whose MHC region has been humanized, comprising the following steps:

[0090] (1) using the vector set described in any one of [1] to

[21] , to prepare a cell comprising a chromosome in which the MHC region of a non-human mammal chromosome is replaced with the MHC region of a human chromosome; and

[0091] (2) A step of obtaining a non-human mammal whose MHC region has been humanized from the cells obtained in (1).

[0092]

[29] The method according to

[28] , wherein:

[0093] Non-human mammals are rodents.

[0094]

[30] The method according to

[29] , wherein:

[0095] Rodents are mice,

[0096] A method for producing a non-human mammal having a humanized MHC region, comprising the step of transplanting a chromosome having a humanized MHC region into a pseudo-parent of a non-human mammal to produce a non-human mammal having a chromosome having a humanized MHC region.

[0097]

[31] A method for producing a non-human mammal whose MHC region has been humanized, comprising:

[0098] The process of transplanting the cells described in

[22] into a pseudo-parent of a non-human mammal to produce a non-human mammal having a chromosome in which the MHC region has been humanized.

[0099]

[32] The method according to

[31] , wherein:

[0100] Non-human mammals are rodents.

[0101]

[33] The method according to

[32] , wherein:

[0102] The rodent is a mouse.

[0103] Effects of the Invention

[0104] According to the present invention, non-human animals having chromosomes obtained by replacing non-human MHC with human MHC can be produced, thereby enabling the development of drugs (such as tumor immunotherapy drugs) and reagents that utilize the human immune system using non-human animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] [ Figure 1 ] is a schematic diagram showing the recombination for replacing non-human MHC with human MHC in the present invention. FIG is a schematic diagram when the non-human animal is a mouse.

[0106] [ Figure 2 ] is a schematic diagram showing the MHC region in human chromosomes.

[0107] [ Figure 3 ] is a diagram showing the target area when a genome editing targeting vector is used.

[0108] [ Figure 4 ] is a schematic diagram of the method for preparing mice with humanized MHC regions.

[0109] [ Figure 5 ] is a diagram showing the structure of the targeting vector. (A) The structure of TV3R-dHLA. (B) The structure of TV4-dH2. (C) The structure of TV-Tl1 / 2-BS. The positions of the homology arms used in the targeting vector on the genome are shown in the figure.

[0110] [ Figure 6 ] are diagrams showing the isolation of A9-MRC5 fusion cell clones that retain human chromosome 6. (A) Genomic DNA was prepared from the isolated A9-MRC5 fusion cell clones (hChA9#1, 5, 13, 17), and the presence of sequences on human chromosome 6 was analyzed by genomic PCR. (B) The presence of human chromosome 6 in hChA9#5 was analyzed by FISH. Red: Human chromosome 6 centromere-specific probe.

[0111] [ Figure 7 ] are diagrams showing the targeting of TV3R. (A) TV3R-dHLA targeting was performed using A9-MRC5 fusion cell cloning, and genomic PCR was used to analyze whether hygromycin-resistant clones were isolated and recombinantly introduced. * is a TV3RhChA9#5-45(128) clone. (B) Recloned from TV3RhChA9#5-45(128) and analyzed using genomic PCR. (C) TV3RhChA9#5-45a was analyzed using FISH. Red: Human chromosome 6 centromere-specific probe.

[0112] [ Figure 8 ] are diagrams showing the targeting of TV4. (A) Analysis of the targeting of TV4-dH2 by genomic PCR. * indicates the clone of TV4RENK A#17. (B) DAPI staining confirmed that the number of chromosomes of TV4RENK A#17 was 40.

[0113] [ Fig. 9 ] is a diagram showing the isolation of TV3-4RENKA clones. FISH analysis was performed on TV3-4RENKA#17 / 5-45a-3 cells into which modified human chromosome 6 had been introduced. The arrow indicates the introduced modified human chromosome 6. The number of chromosomes was confirmed to be 41. Red: mouse Cot-1 (probe for mouse genome sequence), purple: RP11-54H13 (probe in the human MHC region)

[0114] [ Fig.10] is a diagram showing the separation of TV3-4RENKATl3 / 4 clones. (A) Recombinant translocation based on Cre / loxP. (B) Confirmation of translocation based on genomic PCR. * is TV3-4RENKATl3 / 4#17-1. (C) FISH analysis was performed on TV3-4RENKATl3 / 4#17-1. The numbers in the FISH image correspond to the numbers of the translocated chromosomes in the schematic diagram. Red: human Cot-1 (probe of human genome sequence), green: mouse Cot-1 (probe of mouse genome sequence) (D) FISH analysis was performed on TV3-4RENKATl3 / 4#17-1. The numbers in the FISH image correspond to the numbers of the translocated chromosomes in the schematic diagram. Red: RP11-54H13 (probe in the human MHC region), Green: RP23-147G23 (probe on the centromere side of the mouse chromosome 17 MHC region), Purple: RP23-119G24 (probe on the telomeric side of the mouse chromosome 17 MHC region).

[0115] [ Fig.11 ] is a diagram showing the separation of TV3-4RENKATlb clones. (A) Chromosomal translocation was analyzed by genomic PCR. * is TV3-4RENKATlb#38 clone. (B) TV3-4REN KATlb#38 was analyzed by FISH. The numbers in the FISH image correspond to the numbers of the translocated chromosomes in the schematic diagram. Red: RP23-147G23 (probe on the centromere side of the MHC region of mouse chromosome 17), green: human Cot-1 (probe for human genomic sequence), purple: RP23-306H20 (probe on the telomere side of the MHC region of mouse chromosome 17).

[0116] [ Fig.12 ] is a diagram showing the separation of TV3-4RENKATlbdh6 clones. (A) Sorting of TV3-4RENK ATlbdh6. (B) FISH analysis was performed on TV3-4RENKATlbdh6#3. The numbers in the FIS H image correspond to the numbers of the translocation chromosomes in the schematic diagram. The number of chromosomes was confirmed to be 40. Red: RP23-147G23 (probe on the centromere side of the MHC region of mouse chromosome 17), green: human Cot-1 (probe for human genome sequence), purple: RP23-361C19 (probe on the telomere side of the MHC region of mouse chromosome 17). (C) The results of analyzing the HLA haplotype of TV3-4RENKATlbdh6#3 by whole genome sequencing.

[0117] [ Fig.13] are diagrams showing MHC humanized chimeric mice. (A) Appearance of TV3-4RENKATlbdh6#3 derived chimeric mice. (B) Structure of chromosome 17 of MHC humanized mice. An EGFP expression cassette was inserted at the distal side of the MHC region. (C) Expression of EGFP in testicular cells of TV3-4RENKATlbdh6#3 derived chimeric mice was analyzed by flow cytometry.

[0118] [ Fig.14 ] is a diagram showing the preparation of MHC humanized (heterogeneous) mice. (A) MHC humanized (heterogeneous) cells maintain MHC humanized mouse chromosome 17 with an EGFP expression cassette inserted at the distal side of the MHC region. (B) Individuals produced by microinsemination of testicular cells from TV3-4RENKATlbdh6#3-derived chimeric mice. Left: Bright field image. Right: Detection of EGFP fluorescence. (C) Genomes were prepared from offspring obtained by microinsemination, and PCR was used to analyze whether there were sequences in the human MHC region. Offspring with individual numbers 7 and 12 were EGFP-negative.

[0119] [ Fig.15 ] is a diagram showing the preparation of MHC humanized (heterogeneous) mice from XO ES cells. (A) Chimeric mice prepared from TV3-4XOTlbdh6#11-1-4-57-3 clone. Arrow: 100% chimeric mouse. (B) Offspring of XO chimeric mice (female) and C57BL / 6 (male). As long as XO ES cells are not reproduced in a reproductive series, black-haired individuals will not be born. (C) Genomic typing method using the DAXX locus present in the MHC region as a target. Three primers 1 to 3 are used to determine whether the MHC region is of human or mouse origin. The red arrow indicates the position of the primer designed for the portion with the same sequence in humans and mice. The black ones are human or mouse specific primers. (D) An example of genomic typing of offspring obtained by mating XO chimeric mice (female) and C57BL / 6 (male). W: wild-type mouse, H: MHC humanized (heterogeneous) mouse.

[0120] [ Fig.16 ] is a diagram showing the results of the analysis of the expression of human MHC proteins in MHC humanized (heterogeneous) mice. (A) The expression of human MHC proteins in spleen cells was analyzed by Western blotting. Wt, spleen cells from wild-type mice; Het, spleen cells from MHC humanized (heterogeneous) mice. (B) The surface expression of human MHC proteins in spleen cells was analyzed by flow cytometry. Wt, spleen cells from wild-type mice; Het, spleen cells from MHC humanized (heterogeneous) mice. DETAILED DESCRIPTION

[0121] 1. Summary

[0122] The present invention relates to a targeting vector for constructing a non-human chromosome (MHC humanized chromosome) obtained by replacing a non-human MHC region with a human MHC region, a non-human mammal having the MHC humanized chromosome, and a method for producing the same.

[0123] If a non-human animal with a chromosome containing the human MHC region is prepared, the gene that specifies the MHC (MHC gene) can be expressed by an endogenous promoter. Therefore, it is possible to reconstruct the human immune system close to physiological conditions, which is useful for drug development and research on human immune responses.

[0124] However, the length of the human MHC region is approximately 3.4 Mb, and therefore the entire MHC region cannot be introduced, particularly by transgenic methods using bacterial artificial chromosomes (BAC), etc. In addition, the gene density in the MHC region is very high, and there are many genes other than MHC genes.

[0125] Therefore, in the present invention, an attempt was made to replace a non-human MHC region with a human MHC region by utilizing chromosome engineering to produce a non-human animal in which the MHC region has been humanized.

[0126] In the present invention, the outline for replacing the MHC region of a non-human mammal with the human MHC region is as follows: Figure 1 shown.

[0127] exist Figure 1 In the figure, (a) is a schematic diagram of human chromosome 6 and non-human chromosome before replacement, and (c) is a schematic diagram of the chromosome after replacement of the MHC region. For the replacement of the MHC region, a two-stage recombination of translocation recombination from (a) to (b1) and a translocation recombination from (b1) to (c) (this is referred to as "two-stage recombination 1"), or a two-stage recombination of translocation recombination from (a) to (b2) and a translocation recombination from (b2) to (c) (this is referred to as "two-stage recombination 2"). The order of recombination in the present invention is arbitrary, and it can be two-stage recombination 1 or two-stage recombination 2.

[0128] Figure 1 In the MHC region that is the target of translocation recombination (also referred to as "replacement" or "recombination" in this specification), the telomere side is referred to as the distal side, and the centromere side is referred to as the proximal side. Furthermore, an arbitrary position that is the boundary of recombination from the human MHC region to the distal side, that is, from the human MHC region to the distal end, is referred to as P1, and an arbitrary position that is the boundary of recombination from the non-human MHC region to the distal side, that is, from the non-human MHC region to the distal end and corresponds to P1 is referred to as p1. In addition, in Figure 1 In the embodiment, an arbitrary position from the human MHC region to be the object of recombination to the proximal side, that is, between the human MHC region and the centromere, which is the boundary of recombination, is set as P2, and an arbitrary position from the non-human MHC region to the proximal side, that is, between the non-human MHC region and the centromere, which is the boundary of recombination and corresponds to P2 is set as p2.

[0129] At this time, in the two-stage recombination 1, the recombination from (a) to (b1) is the first recombination, which is the recombination of the region from P1 to the distal end (telomere) and the region from p1 to the distal end (telomere). And the recombination from (b1) to (c) is the second recombination, which is the recombination of the region from P2 to the distal end (telomere) and the region from p2 to the distal end (telomere).

[0130] On the other hand, in 2-stage reorganization 2, Figure 1 In the above, the recombination between the region from the centromere side (P2) to the distal end of the human MHC region to be the object of recombination and the region from the centromere side (p2) to the distal end of the non-human MHC region is referred to as the “first recombination” ( Figure 1 (b2)). In this stage, the recombination between the region from P1 to the distal end of the human chromosome and the region from P1 to the distal end of the non-human chromosome does not occur. Then, the recombination between the region from P1 to the distal end of the human chromosome and the region from P1 to the distal end of the non-human chromosome is called "secondary recombination."

[0131] After the completion of the recombination, cells such as ES cells that have undergone humanization of the MHC are produced, which can be transplanted into pseudo-parents to produce chimeric non-human animals. It should be noted that after the completion of the recombination, it is preferred to remove the remaining human chromosomes in the cell. The human chromosomes can be removed by subculturing in the absence of a selection agent. In addition, the chromosomes in non-human mammals are diploid, and in the chromosomes of the non-human mammals after the recombination is completed in the present invention, the form of the chromosomes obtained by replacing the MHC region with the MHC region of the human chromosome can be homologous (two humanized chromosomes) or heterologous (one humanized chromosome).

[0132] Therefore, MHC humanized heterologous animals can be produced by natural mating or microinsemination, and MHC humanized homologous animals can be produced by natural mating or microinsemination. It should be noted that "animal" in this specification means non-human mammals unless otherwise specified.

[0133] Here, human MHC is the same molecule as HLA, and the genes that determine this molecule are arranged in tandem on chromosome 6. The MHC region of non-human mammals varies depending on the animal, for example, it is located on chromosome 17 in mice, chromosome 20 in rats, chromosome 12 in rabbits, and chromosome 7 in pigs.

[0134] It should be noted that when describing a gene encoding MHC in a gene region, it may be described as "MHC region". In addition, the MHC region of mice is present on the long arm, so for example, Figure 1 When the mouse chromosome is represented as a non-human chromosome, the direction of the long arm and the short arm of the mouse is opposite to that of the long arm and the short arm of the human.

[0135] The MHC region to be recombined may be the entire MHC region or a part of it. It may be a classical class I molecule or a non-classical class I molecule in class I. Among classical class I molecules, there are three types in humans, namely HLA-A, HLA-B, and HLA-C, and among non-classical class I molecules, there are HLA-E, HLA-F, HLA-G, MICA, and MICB.

[0136] Human class II genes are located in the human MHC class II region, and the loci of DR, DQ, and DP encode the main products of this region. It should be noted that mouse H2-M2 and H2-M3 also exist in mouse MHC, and in the present invention, these MHCs may not be included in the target region for replacement. The class III region contains various genes other than MHC, and in humans, it is the region on the distal side of HLA-DRA and the proximal side of MICB.

[0137] For example Figure 2 It is a schematic diagram representing a human MHC region. The MHC region that becomes the object of substitution may be only a Class I region, only a Class II region, only a Class III region, or a combination of these regions. In addition, the Class I region may be a region containing a classical Class I MHC gene, a region containing a non-classical Class I MHC gene, or a region of both. Therefore, in the present invention, the regions constituting each level may be, for example, any one of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, MICA, and MICB or a region of a combination of two or more in Class I, and any one of HLA-DP, HLA-DQ, HLA-DR, HLA-DO, and HLA-DM or a region of a combination of two or more in Class II. Various genes other than MHC are included in the Class III region, which is a region of the distal side of HLA-DRA and the proximal side of MICB in humans.

[0138] Here, the MHC region to be the target of recombination may be the entire region or a part of the region as described above. Therefore, by selecting the MHC region to be the target, there is a case where P1, p1, P2, and p2 are located in an MHC region that is not selected.

[0139] In the present invention, in the case of the human MHC class I region, preferably, the region including the entirety of MICB to HLA-F ( Figure 2 In the case of the class II region, the region including the entirety of HLA-DP to HLA-DR ( Figure 2 The class III region is the region on the distal side of HLA-DR A and the proximal side of MICB ( Figure 2 In one embodiment of the present invention, it is further preferred to replace Figure 2 The MHC region represented by Figure 2 The area represented by L4).

[0140] 2. Targeting vector

[0141] (1) The first reorganization in 2-stage reorganization 1 and the second reorganization in 2-stage reorganization 2

[0142] In the present invention, the vector used to cause recombination is called a targeting vector. The targeting vector used herein is not limited to the vector causing the recombination, and may be a vector using a recombinase recognition sequence and a recombinase, or a vector using a genome editing tool. As required, a genome editing tool may be used as a tool for cutting at the positions of P1 and p1. When using a genome editing tool, the chromosome may be cut at the target position, so that subsequent recombination can be easily caused.

[0143] <Recombination using recombinase>

[0144] With regard to the reorganization utilizing recombinase, for example, utilize the recombinase recognition sequence that is called loxP and the recombinase that is called Cre.The specific recombination system of Cre / loxP is well-known.Cre enzyme is the protein that is made up of 343 amino acids from P1 phage source, can recognize the specific base sequence of 34bp that is called loxP site, carries out site-specific recombination.The loxP site can be divided into 3 parts of 13 bases, 8 bases and 13 bases, and the sequence of 13 bases is complementary inverted repeat sequence structure.On the other hand, the sequence of 8 bases plays the role of determining the direction of loxP site.It is well-known that there is the mutant loxP that is called Lox511, lox2272, and these recombinase recognition sequences can also be utilized.

[0145] In another embodiment of the present invention, as other examples of site-specific recombination systems based on site-specific recombinases, in addition to the Cre / loxP system, the Flp / FRT system derived from the yeast plasmid 2μ, the PhiC31 integrase system derived from the bacteriophage PhiC31, the Bxb1 integrase system derived from the bacteriophage Bxb1, etc. can also be used.

[0146] exist Figure 1 In the above, the targeting vector used for recombination from (a) to (b1) or recombination from (b2) to (c) is a combination of the following targeting vector 1 and targeting vector 2 or the following targeting vector 3. The combination of targeting vector 1 and targeting vector 2 is a vector that causes recombination using a recombinase recognition sequence and a recombinase, and targeting vector 3 is a vector that causes recombination using a genome editing tool.

[0147] (i) Targeting vector 1

[0148] The targeting vector 1 is introduced into an arbitrary position P1 on the distal side of the MHC (human MHC) region that exists in the short arm of human chromosome 6 and becomes the object of recombination in human chromosome 6. The targeting vector 1 causes translocation recombination between the region from the P1 to the distal end and the region from the distal side of the non-human MHC region that becomes the object of recombination in the chromosome of a non-human mammal, corresponding to the position p1 to the distal end of the P1. In addition, the targeting vector 1 includes a gene cassette 1, which includes a recombinase recognition sequence, a partial sequence of a drug resistance gene 1 for drug screening reconstructed during translocation recombination, and a drug resistance gene 2 for drug screening after being targeted.

[0149] For the construct contained in the gene cassette 1, for example, the recombinase recognition sequence can be set to lo x P, the drug resistance gene 1 can be set to a neomycin resistance gene, and the drug resistance gene 2 can be set to a hygromycin resistance gene.

[0150] Here, "correspondence" refers to the term for the positional relationship of the position where the recombination occurs. That is, the P1, p1, P2, and p2 refer to markers showing the positional relationship of the distal side or the proximal side from the MHC region that is the object of recombination. Therefore, it is not required that the genes adjacent to P1 and p1 or P2 and p2 are the same. Depending on the type of mammal, there is a case where the MHC region is inverted compared to the direction of the human MHC region. In this case, with respect to the loci on both sides of the position (P1) where the targeting vector is introduced, the human loci are between the MOG locus and the GABBR1 locus (refer to Examples and Figure 5 ), the p1 of the animal in which the MHC region becomes inverted can be, for example, between the Kifc1 locus and the Daxx locus.

[0151] Figure 5Schematic diagram showing the structure of the targeting vector used in the examples of the present invention. For ease of explanation, the targeting vector in which the mouse MHC region is replaced with the human MHC region is described as an example.

[0152] exist Figure 5 In A, "TV3R" represents the gene box 1. The recombinase recognition sequence is a loxP sequence, etc., which is indicated by the horizontal "▲" in the figure. As for the partial sequence of the drug resistance gene 1 for drug screening reconstructed during translocation recombination (the given partial sequence of the drug resistance gene 1 used in the targeting vector 1), "3'neo." is used in the present invention. The drug resistance gene in this case is the neomycin resistance gene. The drug resistance gene 2 for drug screening after targeting is expressed as "Hyg," and the drug resistance gene in this case is the hygromycin resistance gene.

[0153] Furthermore, the gene cassette 1 prepared in the example of the present invention comprises a sequence represented by the following formula (1).

[0154] BGHpA-3'Neo-SA-loxP-EF1-Hyg-P2A-mRuby2-RGpA(1)

[0155] In formula (1), BGHpA represents a poly A tailing signal sequence derived from bovine growth hormone, 3'Neo represents a sequence of a portion of the 3' end of the neomycin resistance gene, SA represents a splice acceptor sequence, loxP represents a recombinase recognition sequence, EF1 represents a promoter sequence derived from human elongation factor 1α, Hyg represents a hygromycin resistance gene, P2A represents a 2A sequence derived from porcine teschovirus, mRuby2 represents a red fluorescent gene, and RGpA represents a poly A tailing signal sequence derived from rabbit β-globin. However, in the present invention, an Flp recombinase recognition sequence (FRT) may be added to the box 1 represented by (1).

[0156] "TV3R-dHLA" is a targeting vector 1 in which sequences identical to those of the regions on both sides of the site (P1) where TV3R is introduced are linked to both sides of TV3R.

[0157] Figure 5 In A, "PX458a-dHLACR1" is a genome editing vector, which artificially cuts off an arbitrary position P1 of the chromosome in order to facilitate the recombination of the targeting vector 1. The genome editing tool used for the genome editing vector can use any known tool. For example, tools using zinc finger ribonucleases (ZFNs), tools using transcription activator nucleases (TALENs), tools using CRISPR-Cas9, etc. can be cited.

[0158] PX458a-dHLACR1 is a vector using CRISPR-Cas9, and is composed of, for example, a guide RNA sequence, a Cas9 sequence, and a gene sequence of a fluorescent protein Ametrine.

[0159] Figure 1 (d) shows an enlarged view of the region from the vicinity of the MHC region to the distal end. In the embodiment of the present invention, the targeting vector 1 is designed to be introduced between the MOG locus and the GABBR1 locus in the human chromosome. Figure 5 A).

[0160] (ii) Targeting vector 2

[0161] The targeting vector 2 is introduced into the chromosome of a non-human mammal having an MHC region, into a position p1 corresponding to the above-mentioned P1, which is distal to the non-human MHC region to be the target of recombination.

[0162] In the mouse of the embodiment, the targeting vector 2 causes translocation recombination between the region from the p1 to the distal end and the region from the P1 to the distal end in the human chromosome. In addition, the targeting vector 2 includes a gene cassette 2, which includes a recombinase recognition sequence, other partial sequences of the drug resistance gene 1 for drug screening reconstructed during translocation recombination (other partial sequences other than the given partial sequence of the drug resistance gene 1 used in the targeting vector 1), and a drug resistance gene 3 for drug screening after being targeted.

[0163] In the construct contained in gene cassette 2, for example, the recombinase recognition sequence can be set to lo x P, the drug resistance gene 1 can be set to a neomycin resistance gene, and the drug resistance gene 3 can be set to a puromycin resistance gene.

[0164] As described above, some animals have inverted MHC regions depending on the species. Even in this case, the targeting carrier 2 can be introduced into the position p1 corresponding to an arbitrary position P1.

[0165] exist Figure 5 In B, "TV4" indicates gene cassette 2. The recombinase recognition sequence is a loxP sequence, etc., which is indicated by the horizontal "▲" in the figure. As for the partial sequence of the drug resistance gene 1 for drug screening reconstructed during translocation recombination, "5'neo." is used in the present invention. The drug resistance gene in this case is the neomycin resistance gene. The drug resistance gene 2 for drug screening after targeting is expressed as "Puro," and the drug resistance gene in this case is the puromycin resistance gene.

[0166] Furthermore, the gene cassette 2 prepared in the example of the present invention comprises a sequence represented by the following formula (2).

[0167] EF1-EGFP-P2A-5'Neo-SD-loxP-SA-T2A-Puro-RGpA(2)

[0168] In formula (2), EF1 represents a promoter sequence derived from human elongation factor 1α, EGFP represents a green fluorescent gene, P2A represents a 2A sequence derived from porcine teschovirus, 5'Neo represents a sequence of a portion of the 5' end of a neomycin resistance gene, SD represents a splice donor sequence, loxP represents a recombinase recognition sequence, SA represents a splice acceptor sequence, T2A represents a 2A sequence derived from East Asian serovar (Thoseaasigna) virus, Puro represents a puromycin resistance gene, and RGpA represents a poly A tailing signal sequence derived from rabbit β-globin. In the present invention, FRT can be added to the box 2 represented by formula (2).

[0169] TV4-dH2 is a targeting vector 2 in which sequences identical to the sequences of the regions on both sides of the position (p1) into which TV4 is introduced are linked to both sides of TV4.

[0170] Figure 5 In B, "PX458a-dH2CR1" is a genome editing vector that artificially cuts off an arbitrary position p1 of the chromosome in order to facilitate the recombination of the targeting vector 2. The genome editing tool used for the genome editing vector is the same as that described above. PX458a-dH2CR1 is a vector using CRISPR-Cas9, for example, composed of a guide RNA sequence, a Cas9 sequence, and a gene sequence of the fluorescent protein Ametrine.

[0171] and Figure 1 (d) Similarly, in the embodiment of the present invention, the targeting vector 2 is designed to be introduced between the Mog locus and the Gabbr1 locus in the non-human chromosome. Figure 5 B)

[0172] <Recombination using genome editing vector>

[0173] (iii) Targeting vector 3

[0174] In another embodiment of the present invention, in order to perform the first recombination, a genome editing tool can be used instead of the recombination using the recombinase. In the case of the targeting vector 3, a translocation recombination is caused between a region from a position P1 to a distal end of a human MHC region that is a target of the recombination and is cut by the genome editing tool, and a region from a position p1 to a distal end of a non-human MHC region that is a distal end of a chromosome of a non-human mammal having MHC and corresponds to the P1.

[0175] In this case, replacement of the MHC region can be utilized Figure 1 Translocation recombination from (a) to (b1) and from (b1) to (c) (2-stage recombination 1) or translocation recombination from (a) to (b2) and from (b2) to (c) (2-stage recombination 2).

[0176] The targeting vector 3 comprises: a sequence identical to the sequence of a portion of the region between the P1 and the human MHC region (the region is called "R1a"), a drug resistance gene 4 for drug screening after targeting, and a sequence identical to the sequence of a portion of the region on the distal side of the p1 (the region is called "R2b"), or the targeting vector 3 comprises: a sequence identical to the sequence of a portion of the region on the distal side of the p1 (the region is called "R1b"), a drug resistance gene 4 for drug screening after targeting, and a sequence identical to the sequence of a portion of the region between the p1 and the non-human MHC region (the region is called "R2a").

[0177] The positional relationship among R1a, R1b, R2a and R2b is as follows Figure 3 It should be noted that the regions of R1a, R1b, R2a and R2b may be any region from the perspective of P1 and p1, and are preferably regions near P1 and p1.

[0178] The targeting vector 3 used in the present invention can be composed of, for example, an R2a sequence, a CAG promoter sequence, a blasticidin resistance gene, a rabbit β-globin-derived poly A tailing signal sequence, and an R1b sequence.

[0179] (2) The second reorganization in 2-stage reorganization 1 or the first reorganization in 2-stage reorganization 2

[0180] In the present invention, the second recombination in 2-stage recombination 1 and the first recombination in 2-stage recombination 2 will be caused, that is, Figure 1 The reorganization from (b1) to (c) or Figure 1 The recombinant vector from (a) to (b2) is called targeting vector 4.

[0181] The recombination in this section can be carried out in the same manner as described above using a targeting vector using a recombinase. According to preliminary experiments conducted by the inventors, a targeting vector using a genome editing tool more efficiently induces recombination than a targeting vector using a recombinase, and is also effective for the subsequent production of non-human mammals.

[0182] Therefore, in the present invention, the second recombination in the two-stage recombination 1 or the first recombination in the two-stage recombination 2 is performed by using a genome editing tool.

[0183] (iv) Targeting vector 4

[0184] Regarding the targeting vector 4, in the case of the two-stage recombination 1, the chromosome ( Figure 1 In (b1)), translocation recombination is caused between a region from an arbitrary position P2 to the distal end on the proximal side of the human MHC region to be recombinated and a target to be cut by a genome editing tool, and a region from a position p2 to the distal end on the proximal side of the non-human MHC region corresponding to the aforementioned P2 in a chromosome of a non-human mammal in which a non-human MHC is present.

[0185] In addition, regarding the use of targeting vector 4, in the case of two-stage recombination 2, targeting vector 4 can be used first and then targeting vectors 1 to 3. Figure 1 For the recombination represented by (a) to (c), recombination from P1 and p1 to the distal end can be performed first, and then recombination from P2 and p2 to the distal end can be performed ( Figure 1 (a), (b1) and (c) in sequence), or recombination from P2 and p2 to the distal end may be performed first, followed by recombination from P1 and p1 to the distal end ( Figure 1 (a), (b2) and (c) in that order).

[0186] The targeting vector 4 comprises: a sequence identical to the sequence of a portion of the region R3a on the proximal side of the P2 (between P2 and the centromere), a drug resistance gene 5 for drug screening after targeting (e.g., a blasticidin resistance gene), and a sequence identical to the sequence of a portion of the region R4b between the p2 and the non-human MHC region; or, the targeting vector 4 comprises: a sequence identical to the sequence of a portion of the region R3b between the P2 and the human MHC region, a drug resistance gene 5 for drug screening after targeting, and a sequence identical to the sequence of a portion of the region R4a from the p2 to the proximal side (between p2 and the centromere).

[0187] The positional relationship among R3a, R3b, R4a and R4b is as follows Figure 3 It should be noted that the regions of R3a, R3b, R4a and R4b may be any region when viewed from P2 and p2, and are preferably regions near P2 and p2.

[0188] The targeting vector 4 used in the present invention can be composed of, for example, an R3a sequence, a CAG promoter sequence, a blasticidin resistance gene, a rabbit β-globin-derived poly A tailing signal sequence, and an R4b sequence.

[0189] Figure 1(e) shows an enlarged view of the region from the vicinity of the MHC region to the distal end. In the embodiment of the present invention, the targeting vector 4 is designed so as to cause recombination between the KIFC1 locus and the DAXX locus in the human chromosome and between the Kifc1 locus and the Daxx locus in the non-human chromosome. Figure 5 C). Targeting vector 4 contains a blasticidin (BS) resistance gene.

[0190] exist Figure 5 In C, "PX458.1a-pHLACR2" is a genome editing vector that artificially cuts off an arbitrary position P2 of the chromosome in order to facilitate translocation recombination by targeting vector 4. PX458.1a-pHLACR2 is a vector using CRISPR-Cas9, and is composed of, for example, a guide RNA sequence, a Cas9 sequence, and a gene sequence of a fluorescent protein Ametrine.

[0191] Similar to PX458.1a-pHLACR2, "PX458.1a-pH2CR1" is a genome editing vector that artificially cuts off an arbitrary position p2 of the chromosome in order to facilitate translocation recombination by targeting vector 4. PX458.1a-pH2CR1 is a vector using CRISPR-Cas9, and is composed of, for example, a guide RNA sequence, a Cas9 sequence, and a gene sequence of a fluorescent protein Ametrine.

[0192] <Drug resistance genes for drug screening>

[0193] In the present invention, as described above, drug resistance genes 1 to 5 for drug screening are used in the targeting vector. The combination of each gene of drug resistance genes 1 to 5 is arbitrary and is appropriately selected so that target drug screening can be performed.

[0194] Examples of genes used in drug-resistant genes 1 to 5 include, but are not limited to, neomycin resistance gene, hygromycin resistance gene, puromycin resistance gene, and blasticidin resistance gene.

[0195] <Fluorescent gene for recombination or screening confirmation>

[0196] In the present invention, a given fluorescent gene may be included in the targeting vector in order to confirm the chromosome introduction, the maintenance of the introduced chromosome, or whether the drug screening has been performed according to the purpose. The type of fluorescent gene may be arbitrarily selected as long as the confirmation operation can be performed in each step, and the type of fluorescent gene may be changed for each vector.

[0197] Examples of the fluorescent gene include a green fluorescent gene (GFP, EGFP, etc.), a red fluorescent gene, and a yellow fluorescent gene.

[0198] 3. Cells

[0199] In order to create a non-human mammal in which MHC has been humanized, it is necessary to replace the endogenous MHC genes in the cells of the non-human mammal with human MHC genes.

[0200] In the present invention, the general homologous recombination method or genome editing technology as described above can be used.

[0201] The cells are not particularly limited as long as they are animal cells, and examples thereof include ES cells, sperm stem cells, and fibroblasts, among which ES cells are preferred.

[0202] Examples of the cell culture medium include GMEM medium (Glasgow's Minimal Essential Medium), DMEM (Double's Modified Eagle Medium), RPMI1640 medium, etc. Commonly used animal cell culture additives such as fetal bovine serum (FBS), non-essential amino acids, antibiotics (e.g., penicillin, streptomycin, etc.), growth factors / cytokines (epidermal growth factor, fibroblast growth factor, leukemia inhibitory factor, etc.) can be appropriately added to the culture medium according to the cell type.

[0203] After the cells are cultured for a given period, the cells are recovered by hatching with a culture medium containing trypsin. With respect to the recovered cells, multiple subcultures can be performed as needed in the presence or absence of feeder cells. With respect to the confirmation that the cells obtained by culture are target cells, their marker genes can be used as indicators. In the case where the cells are ES cells, for example, Oct3 / 4, alkaline phosphatase, Nanog, etc. can be used as indicators, and any method such as RT-PCR, Western blotting can be used for detection. In addition, ES cells can be judged by cell group morphology.

[0204] 5. Preparation of Non-human Mammals

[0205] The production of non-human mammals can be carried out by standard methods such as somatic cell nuclear transfer and microinsemination using sperm stem cells, in addition to the production of chimera animals using ES cells.

[0206] The non-human mammals to be prepared are not particularly limited, and examples thereof include rodents, livestock, primates, etc. Examples of rodents include mice, rats, guinea pigs, hamsters, etc. Examples of livestock include cattle, horses, pigs, and sheep, and examples of primates include Japanese macaques and common marmosets. In addition, pets or experimental animals such as dogs, cats, monkeys, and rabbits may also be used.

[0207] In the present invention, rodents are preferred, and mice are particularly preferred.

[0208] In the case of chimeric animals, first, the established ES cells are aggregated with an 8-cell stage embryo or injected into a blastodisc. The embryo thus prepared is called a chimeric embryo, and the chimeric animal is prepared by transplanting the chimeric embryo into the uterus of a pseudopregnant parent and giving birth.

[0209] Here, the term "embryo" refers to an individual at the stage from fertilization to birth, including 2-cell embryos, 4-cell embryos, 8-cell embryos, morulae, blastocysts, and the like.

[0210] In the following, for the sake of convenience, ES cells are used as an example of cells.

[0211] As a method for preparing aggregates using ES cells and embryos, known methods such as microinjection and agglutination can be used.

[0212] When adopting microinjection, ES cells are injected into the embryos recovered to prepare cell aggregates. In addition, when adopting agglutination, ES cells are sprinkled on normal embryos from which the zona pellucida has been removed to agglutinate. The ES cells used herein are not particularly limited, and examples thereof include ES cells established by mixing the Y chromosome-shedding strain XO of TT2 cells from C57BL / 6 source RENKA, C57BL / 6 and F1 of CBA, and F1 of C57BL / 6 and DBA / sCrSlc.

[0213] On the other hand, pseudo-pregnant female animals used as pseudo-parents can be obtained by mating female animals with normal sexual cycles with male animals castrated by vasectomy, etc. For the pseudo-pregnant animals produced, chimeric embryos prepared by the above method can be transplanted into the uterus, and then chimeric animals can be prepared by giving birth.

[0214] From such chimera animals, select the animal from which the ES cell transplant embryo originated. Make the selected chimera animal mate with an animal of a close relative. And, it can be determined that the ES cell has been introduced into the chimera animal reproductive series by the appearance of the fur color of the animal derived from the ES cell in the offspring animals born.

[0215] Whether or not the offspring animals born retain the humanized MHC gene can be determined by cutting the DNA with restriction endonucleases to detect the presence of DNA fragments of a target size or by analyzing the DNA using the PCR method.

[0216] Example

[0217] Hereinafter, the present invention will be described in more detail by way of examples, but the scope of the present invention is not limited by these examples.

[0218] 1. Preparation of Targeting Vector

[0219] <Method>

[0220] method

[0221] Preparation of TV3R-dHLA

[0222] pEF-GFP (Addgene plasmid #11154; http: / / n2t.net / addgene:11154; RRID:Addgene_11154) was digested with restriction enzymes EcoRI-HF (New England BioLabs) and NotI-HF (New England BioLabs), and the hygromycin resistance gene was cloned downstream of the promoter sequence derived from human elongation factor 1α using In-Fusion (registered trademark) HD ​​Cloning Kit (Clontech) according to the instructions provided therewith, thereby preparing pEF-Hyg (SEQ ID NO. 1). Next, the upstream side of the promoter sequence derived from human elongation factor 1α encoding pEF-Hyg was digested with restriction enzyme SalI, and the synthetically prepared DNA fragment FRT-BGHpA-3'Neo-SA-loxP (Thermo Fisher Scientific) (SEQ ID NO. 2) was cloned using In-Fusion (registered trademark) HD ​​Cloning Kit, thereby preparing TV3.

[0223] Next, the sequence including the 5' identical region and the 3' identical region of the targeting vector TV3R-dHLA was amplified using the genome of MRC5 cells as a template and the following primer set by PrimeSTAR (registered trademark) GXL DNA Polymerase (Takara) according to the attached instruction manual.

[0224]

[0225] Furthermore, the entire same region was amplified by the Nested-PCR method using the following primer set, and cloned using pGEM (registered trademark)-T Easy Vector (Promega) to prepare pGEMTe-dHLA.

[0226]

[0227] Next, pGEMTe-dHLA was amplified using the following primer set and PrimeSTAR (registered trademark) GXL DNA Polymerase, TV3 was restriction endonuclease digested with SalI-HF (New England BioLabs) and HindIII-HF (New England BioLabs) to prepare FRT-BGHpA-3'Neo-SA-loxP, and the fragment containing the prepared FRT-BGHpA-3'Neo-SA-loxP was cloned using In-Fusion (registered trademark) HD ​​Cloning Kit to prepare TV3-dHLA.

[0228]

[0229] The synthetically prepared DNA fragment P2A-mRuby2 (SEQ ID NO. 9) (Thermo Fisher Scientific) was further amplified using the following primer set and PrimeSTAR (registered trademark) GXL DNA Polymerase, and the amplified product and TV3-dHLA were digested with restriction endonuclease BssHII (New England BioLabs), and then the two were ligated using Ligation-Convenience Kit (Nippon gene) to prepare TV3R-dHLA.

[0230]

[0231] Preparation of TV4-dH2

[0232] pEF-GFP (Addgene plasmid #11154; http: / / n2t.net / addgene:11154; RRID:Addgene_11154) was digested with restriction endonucleases EcoRI-HF and NotI-HF, and the EGFP gene was cloned downstream of the promoter sequence derived from human elongation factor 1α using In-Fusion (registered trademark) HD ​​Cloning Kit according to the instructions provided therewith to prepare pEF-EGFP2 (SEQ ID NO. 12). Next, pEF-EGFP2 was digested with restriction endonucleases BsrGI (New England BioLabs), and the sequence P2A-5'Neo-SD-loxP-SA-T2A-Puro (Thermo Fisher Scientific) (SEQ ID NO. 13) was synthesized as two DNA fragments, and cloned using In-Fusion (registered trademark) HD ​​Cloning Kit to prepare TV4dF.

[0233] TV4dF was digested with restriction endonucleases using SalI-HF, and the following oligonucleotide DNAs obtained by annealing were ligated using a Ligation-Convenience Kit to prepare TV4.

[0234]

[0235] Next, the sequence including the 5' homologous region and the 3' homologous region of the targeting vector TV4-dH2 was amplified using the genome of C57BL / 6 mice as a template using the following primer set and PrimeSTAR (registered trademark) GXL DNA Polymerase according to the attached instruction manual.

[0236]

[0237] Furthermore, the entire same region was amplified by the Nested-PCR method using the following primer set, and cloned using pGEM (registered trademark)-T Easy Vector (Promega) to prepare pGEMTe-dH2.

[0238]

[0239] Next, pGEMTe-dH2 was amplified using the following primer set and PrimeSTAR (registered trademark) GXL DNA Polymerase, TV4 was restriction endonuclease digested with SalI-HF and HindIII-HF, and the fragment containing the prepared P2A-5'Neo-SD-loxP-SA-T2A-Puro was cloned using In-Fusion (registered trademark) HD ​​Cloning Kit to prepare TV4-dH2.

[0240]

[0241] Preparation of TV-TI1 / 2-BS

[0242] Using pGEM (registered trademark)-T Easy Vector as the vector backbone, the Hprt expression vector TV-Tl1 / 2-Hprt (SEQ ID NO.22) that maintains the 5' end identical region and the 3' end identical region used in TV-Tl1 / 2-BS was digested with restriction endonucleases XhoI (New England BioLabs) and XbaI (New England BioLabs), and a DNA fragment (IDT) (SEQ ID NO.23) containing the blasticidin resistance gene was prepared by synthesis, and cloned using In-Fusion (registered trademark) HD ​​Cloning Kit to prepare TV-Tl1 / 2-BS.

[0243] Preparation of genome editing vectors (1)

[0244] The gRNAs of PX458a-dHLACR1 and PX458a-dH2CR1 were designed for the following sequences. The gRNA and Cas9 expression used PX458a (SEQ ID NO. 24) prepared by modifying pSpCas9(BB)-2A-GFP(PX458) (Addgene plasmid#48138; http: / / n2t.net / addgene:48138; RRID:Addgene 48138) and replacing the EGFP gene with the fluorescent gene Ametrine.

[0245]

[0246] Preparation of genome editing vectors (2)

[0247] The gRNAs of PX458.1a-pHLACR2 and PX458.1a-pH2CR1 were designed for the following sequences, and PX458.1a (SEQ ID NO. 27), which was a modified version of the gRNA scaffold sequence of PX458a, was used in the expression of gRNA and Cas9.

[0248]

[0249] Preparation of drug-resistant mouse fetal fibroblasts (MEF)

[0250] In order to culture ES cells on feeder cells during drug selection, drug-resistant MEFs were prepared. First, MEFs were prepared from 14.5-day embryos, and MEFs were infected with a lentivirus expressing a gene (HBPN) linked to hygromycin resistance gene, blasticidin resistance gene, puromycin resistance gene, and neomycin resistance gene through a 2A sequence. In order to improve the proliferation ability of MEFs, HBPN-expressing MEFs were cultured under hypoxic conditions of 5% oxygen / 5% CO2, treated with mitomycin C, and used as drug-resistant MEFs.

[0251] Cloning of hChA9 from MRC5-A9 fusion cells

[0252] MRC5 cells into which the Neo resistance gene was introduced using a lentivirus were fused with A9 cells using GenomONE-CF (Ishihara Sangyo), and Ouabain-resistant / G418-resistant cells were isolated as fused cells. Human cells are Ouabain-sensitive, while mouse cells are Ouabain-insensitive, and fused cells can be selected by adding G418 and Ouabain to the culture medium. Fusion cells that retain human chromosome 6 were confirmed by genomic PCR using the following primer set set in the intergenic region of KIFC1 and DAXX.

[0253]

[0254] In addition, in the detection of human chromosome 6 based on FISH, referring to the report of Jabs et al. (Am. J. Hum. Genet. 41: 374-390, 1987), a DNA fragment artificially synthesized by focusing on a specific sequence at the centromere of human chromosome 6 was used for the preparation of a probe.

[0255] The synthesized sequence is as follows:

[0256]

[0257] Cloning of TV3RhChA9

[0258] For the clone (TV3RhChA9) obtained by targeting TV3R-dHLA to MRC5-A9 fusion cells, PX458a-dHLACR1 and the targeting vector TV3R-dHLA were transfected into hChA9 using PEI max (Polysciences) for preparation. PX458a-dHLACR1 was co-introduced to improve the homologous recombination efficiency of the targeting vector. The expression of the fluorescent gene mRuby2 on TV3R-dHLA was used as an index to sort the vector-introduced cells on the second day of transfection, and the clones of mRuby2 positive hygromycin resistance were isolated. Genomic DNA was prepared from the clones obtained by separation, and the clones that had been recombined and introduced were confirmed by PCR. The primers used for PCR are as follows: The same analysis was also performed in the case of recloning from the clones obtained by separation.

[0259]

[0260] Clone of TV4RENKA

[0261] TV4RENKA is an ES cell clone prepared by transfecting PX458a-dH2CR1 and the targeting vector TV4-dH2 into RENKA using Lipofectamine 3000 (Thermo Fisher Scientific). PX458a-dH2CR1 was co-introduced to improve the homologous recombination efficiency of the targeting vector. EGFP-positive clones were isolated, and clones that had been recombined were confirmed by genomic PCR. Cloning and isolation can be performed similarly for XO and BDF1.

[0262] The primers used for PCR are as follows:

[0263]

[0264] Cloning of TV3-4RENKA

[0265] TV3-4RENKA is an ES cell clone prepared by introducing modified human chromosome 6 from TV3RhChA9 into TV4RENKA using the retro-MMCT method (Suzuki, T. et al. (2016) PloS One, 11(6): e0157187.). mRuby2-positive hygromycin-resistant cells were cloned, and a clone with 41 chromosomes was isolated. FISH analysis was performed using Cy3-labeled mouse Cot-1 and Cy5-labeled RP11-54H13.

[0266] Cloning of TV3-4RENKAT13 / 4

[0267] Cre expression vector was introduced into TV3-4RENKA using Lipofectamine 3000 to induce inter-loxP chromosome translocation. The clone that caused the chromosome translocation was isolated using G418 resistance as an indicator and named "TV3-4RENKAT13 / 4." After recombination was confirmed by genomic PCR, translocation between mouse chromosome 17 and human chromosome 6 was confirmed by FISH analysis.

[0268] The primers used for PCR are as follows:

[0269]

[0270] In the FISH analysis, detection was performed using Cy3-labeled RP11-54H13, DY490-labeled RP23-147G23, and Cy5-labeled RP23-119G24, or Cy3-labeled human Cot-1 and DY490-labeled mouse Cot-1.

[0271] Cloning of TV3-4RENKAT1b

[0272] In the clone of TV3-4RENKAT1b, TV-Tl1 / 2-BS, PX458.1a-pHLACR2 and PX458.1a-pH2CR1 were co-introduced into TV3-4RENKAT13 / 4 using Lipofectamine 3000. Ametrine positive cells were sorted on the second day of transfection to separate the clone of blasticidin resistance. After recombination was confirmed by genomic PCR, displacement in the MHC region was confirmed by FISH analysis.

[0273] The primers used for PCR are as follows:

[0274]

[0275] In the FISH analysis, detection was performed using Cy3-labeled RP11-147G23, DY490-labeled human Cot-1, and Cy5-labeled RP23-306H20.

[0276] Cloning of TV3-4RENKATlbdh6

[0277] In order to separate TV3-4RENKATlbdh6 after remaining human chromosome 6 falls off from TV3-4RENKATlbdh6, sorting mRuby2 negative cells, is inoculated into the separation of clones on feeder cells.In addition, the FISH analysis of 40 clones (39 in the case of XO) of chromosome number carried out by karyotype analysis has confirmed that remaining human chromosome 6 has fallen off.In FISH analysis, people Cot-1 and the RP23-361C19 of Cy5 mark using RP11-147G23, DY490 mark of Cy3 mark have been detected.The separation of TV3-4XOTlbdh6, TV3-4BDF1Tlbdh6 is also implemented by the step identical with TV3-4RENKATlbdh6.

[0278] 2. Preparation of MHC humanized mice

[0279] With regard to MHC humanized chimeric mice, MHC humanized ES cells and ICR-derived embryos were used to prepare by the collective chimera method. MHC humanized mice were separated by testis sperm cells and microinsemination was performed to the egg of B6 / DBA F1 mice or ICR mice and established using the chimeric mice prepared by RENKA-derived MHC humanized ES cells. In addition, in the case of female chimeric mice prepared using XOES cell-derived MHC humanized ES cells, MHC humanized chimeric mice (MHC humanized heterologous mice) were mated with the male mice of C57BL / 6. The establishment of MHC humanized mice was confirmed by the EGFP fluorescence and genomic PCR method of offspring.

[0280] The primer sets used for genomic PCR were as follows.

[0281] DAXX:

[0282]

[0283] MICA:

[0284]

[0285] MOG:

[0286]

[0287] S76:

[0288]

[0289] DAXX (confirmed by 3 primers):

[0290] Human / mouse common primers,

[0291] Mouse specific primers,

[0292] Human specific primers,

[0293] 3. Results and Investigation

[0294] Construction of vectors for replacement of the MHC region

[0295] First, the necessary vectors were constructed under a series of recombination operations. Considering the effect of transposition on the expression of nearby genes, targeting vectors TV3R-dHLA5, TV4-dH2 ( Figure 5 A, B). In addition, in order to improve the efficiency of homologous recombination, CRISPR vectors PX458a-dHLACR1 and PX458a-dH2CR1 were prepared to induce DNA double-strand cuts at various target sites.

[0296] TV3R encodes the loxP sequence necessary for translocation induction, the 3' end sequence of the Neo resistance gene used to confer G418 resistance during translocation recombination, the FRT sequence used to remove sequences in the targeting vector after translocation induction, the hygromycin resistance gene used for drug selection during targeting, and the red fluorescent gene mRuby2 used to detect the presence of chromosomes after targeting.

[0297] On the other hand, TV4 encodes the loxP sequence necessary for translocation induction, the 5' end sequence of the Neo resistance gene for G418 resistance during translocation recombination, the FRT sequence for removing the sequence in the targeting vector after translocation induction, the puromycin resistance gene for drug selection during targeting, and the green fluorescent gene EGFP for detecting the presence of chromosomes after targeting. The TV3R sequence and TV4 sequence are designed to leave the Neo resistance gene reconstructed as an EGFP expression unit in the mouse chromosome 17 after translocation induction, and the MHC humanized mouse chromosome 17-maintaining cells can be selected by drugs and fluorescence.

[0298] Next, a targeting vector TV-T1 1 / 2-BS was prepared for inducing translocation recombination between the DAXX and KIFC1 loci, which have relatively large intergenic sequences in the proximal side of the MHC region of humans and mice, and CRISPR vectors PX458.1a-pHLACR2 and PX458.1a-pH2CR1 ( Figure 5C). A blasticidin resistance gene expression cassette was added to TV-T1 1 / 2-BS, so that the cells into which the recombinant introduction had been carried out were designed to be selected by blasticidin resistance. In addition, a vector design was carried out so that the blasticidin resistance gene was recombinantly introduced on the remaining human chromosome 6 that was finally removed from the ES cells. Thus, no foreign gene expression cassette was introduced into the MHC region proximal side of the MHC humanized mouse chromosome 17, so it is believed that the influence on the gene expression around the humanized MHC region can be minimized.

[0299] Preparation of A9-MRC5 fusion cells

[0300] In order to introduce human chromosome 6 of human normal fibroblast MRC5 cells into mouse ES cells (hereinafter referred to as ES cells), it is necessary to prepare fusion cells of MRC5 cells and mouse fibroblast A9 cells with high chromosome supply capacity. Therefore, first, in order to select the fusion cells for drugs, a neomycin resistance gene was introduced into MRC5 cells using a lentivirus ( Figure 4 , step 1 (circled number 1. After that, the numbers of each step after step 1 are also given circle numbers in the same way as step 1.)). Next, neomycin-resistant MRC5 cells and A9 cells were fused to isolate the fusion cell line hChA9 ( Figure 4 , step 2).

[0301] When the presence of sequences in the human HLA region in the isolated clones was confirmed by genomic PCR, signals were detected in clones #1, 5, and 13 ( Figure 6 A). In addition, in order to confirm the presence of human chromosome 6 in hChA9#5, the centromere sequence-specific probe of human chromosome 6 was used for analysis by the FISH method, and it was confirmed that two human chromosomes 6 were present in the fusion cell clone ( Figure 6 B).

[0302] TV3R's Target Shooting

[0303] The targeting vector TV3R-dHLA was recombined and introduced into the human chromosome 6 of hChA9#5 at the distal side of the MHC region, and the sequence necessary for replacement of the MHC region was introduced ( Figure 4 , step 3).

[0304] As a result, clones that had become hygromycin-resistant were isolated and analyzed by genomic PCR, and multiple cells that were targeted with TV3RhChA9 ( Figure 7A). In order to exclude the possibility of clonal mixing, TV3RhChA9#5-45a(128) was isolated by recloning from TV3RhChA9#5-45(128) ( Figure 7 B). When TV3RhChA9#5-45a(128) was analyzed by FISH, it was confirmed that more than half of the cells retained one human chromosome 6 ( Figure 7 C) Therefore, TV3RhChA9#5-45a(128) was used as a donor cell for modifying human chromosome 6.

[0305] TV4's Target Practice

[0306] By targeting TV4-dH2 at the distal side of the MHC region present on chromosome 17 of B6-derived mouse ES cell RENKA, the sequence required for the exchange of the MHC region was introduced ( Figure 4 , step 4).

[0307] When EGFP-positive clones were analyzed by genomic PCR, multiple clones with recombinant introduction were confirmed ( Figure 8 A). As for EGFP in the TV4 sequence, if it only exists in the MHC humanized allele, it can be used to confirm the reproduction of the reproductive series. Therefore, the clone TV4RENKA#17, which only targets the single allele of chromosome 40, was used for subsequent experiments ( Figure 8 ).

[0308] Introduction of modified human chromosome 6

[0309] The modified human chromosome 6 of TV3RhChA9#5-45a(128) was introduced into TV4RENKA#17( Figure 4 , step 5). Generally, the chromosome introduction efficiency in ES cells is low, and the fusion cells tend to have a lower chromosome supply capacity than A9 cells, so it is considered that the previous chromosome introduction method using polyethylene glycol (PEG-MMCT method) may not be able to introduce chromosomes. Therefore, a high-efficiency chromosome introduction method (retro-MMCT method) using a monotropic envelope protein derived from mouse leukemia virus was used to introduce modified human chromosome 6.

[0310] ES cells that had become hygromycin-resistant and mRuby2-positive were cloned, and chromosome specimens were prepared and karyotype analysis was performed. It was confirmed that the number of chromosomes in normal mouse cells was 40 (39 for XO), and the number of chromosomes in TV3-4RENKA#17 / 5-45a-3 was 41 (40 for XO). In addition, it was confirmed that a subcentromeric chromosome that is a characteristic of human chromosome 6 was retained, so when further FISH analysis was performed, the chromosome contained a human MHC region. Therefore, it is believed that the modified human chromosome 6 ( Fig. 9 ).

[0311] Induction of chromosomal translocation using Cre / loxP

[0312] The recombinase Cre can induce translocation between loxP sequences on chromosomes that have been separated. Therefore, a translocation was induced between the modified human chromosome 6 that maintained TV3-4RENKA#17 / 5-45a-3 and the MHC region distal side of the modified mouse chromosome 17, thereby transiently expressing the recombinase Cre through TV3-4RENKA#17 / 5-45a-3 ( Figure 4 , step 6). When translocation recombination is induced between lox P present in the TV3 sequence and TV4 sequence, in order to reconstruct the Neo resistance gene ( Fig.10 A), and clones that were resistant to G418 were isolated. Among these clones, genomic PCR and FISH confirmed that the number of chromosomes was 41 (40 for XO), so translocation recombination occurred. This clone was designated as "TV3-4RENKA Tl3 / 4#17-1" and used in subsequent experiments ( Fig.10 BD).

[0313] Induction of chromosomal translocation using CRISPR / Cas9

[0314] With regard to the transposition of the proximal side of the MHC region, the plan uses the recombinase Dre to carry out transposition induction in the same manner as the distal side, therefore the ES cell of the recognition sequence rox of Dre that was initially introduced into the proximal side reorganization is prepared.Yet the efficiency of the transposition carried out by Dre is lower, and in addition, with regard to the expression of the HPRT gene that the plan uses as a selective marker, demand improves in the ES cell, so the expression of the HPRT gene that is reconstructed by the recombinant transposition does not have resistance to the selection cultivation based on HAT.Therefore, the modification of No. 17 chromosome of mouse is changed into the importing of TV4 only, stops the Hprt gene destruction of ES cell and prepares TV3-4RENKAT13 / 4#17-1.

[0315] Therefore, CRISPR / Cas9 was used to induce translocation on the proximal side of the MHC region. Specifically, the CRISPR vector that cuts the intergenic region from KIFC1 to DAXX on mouse chromosome 17 and human chromosome 6 and the translocation induction targeting vector TV-Tl1 / 2-BS were introduced into TV3-4RENKA Tl3 / 4#17-1, and clones that were resistant to blasticidin were isolated ( Figure 4 , step 7). Among them, the number of chromosomes in clone TV3-4RENKA Tlb#38 was confirmed by genomic PCR to be 41 (40 for XO), and translocation recombination occurred. In addition, when analyzed by FISH method, it was found that the modified human chromosome 6 was translocated with the modified chromosome 17 instead of the wild-type chromosome 17, so TV3-4RENKA Tlb#38 was used for subsequent experiments ( Fig.11 ).

[0316] Isolation of MHC humanized ES cells

[0317] In order to ensure the undifferentiated nature of the MHC humanized ES cells, the initial plan was to introduce the MHC humanized mouse chromosome 17 prepared so far into ES cells with a small number of passages, thereby deleting the endogenous mouse chromosome 17 and preparing MHC humanized ES cells ( Figure 4 , step 8'). However, when performing this operation, it was found that it was necessary to prepare ES-A9 fusion cells and introduce MHC humanized chromosomes into ES cells. In this process, chromosomal abnormalities occurred frequently, which limited the operation. Therefore, an attempt was made to separate multiple MHC humanized ES clones from TV3-4RENKA Tlb#38 by naturally shedding human chromosomes to prepare chimeric mice ( Figure 4 , step 8).

[0318] As for human chromosomes, it is known that they are easily shed in mouse cells. Therefore, TV3-4RENKATlb#38 was cultured for several days in the absence of a selection agent to induce the shedding of the remaining human chromosome 6. As for the isolation of the remaining human chromosome 6 shedding clone, it was initially planned to use 6-thioguanine, which can select cells with HPRT gene deletion, as a selection agent, but as mentioned above, it is known that the Hprt gene is difficult to use as a selection marker in ES cells.

[0319] Therefore, the expression of the fluorescent gene mRuby2 designed to remain on the remaining human chromosome 6 was used as an indicator, and mRuby2 negative cells were sorted and clones were isolated. As a result, the separation of the TV3-4RENKATlb#38dh6 clone with the remaining human chromosome 6 removed was successfully performed ( Fig.12). Among them, TV3-4RENKA Tlbdh6#3, which has 40 chromosomes (39 for XO) and MHC humanized mouse chromosome 17 confirmed by FISH, was used for subsequent experiments. In addition, whole genome sequencing analysis of TV3-4RENKA Tlbdh6#3 was performed using mouse genome sequences and sequences in the human MHC region as controls, and the haplotype of the introduced human MHC gene group was confirmed ( Fig.12 C).

[0320] Establishment of MHC humanized mice

[0321] The chimeric mice were prepared from TV3-4RENKATlb#38dh6#3, and three mice with a chimeric rate of 50% to 60% were obtained ( Fig.13 A)( Figure 4 , step 9).

[0322] ES-derived cells that maintain the humanized MHC chromosomes were engineered to express EGFP ( Fig.13 B) Therefore, when the contribution rate of MHC humanized ES cells in the testis was analyzed using the expression of EGFP as an indicator, it was found that approximately 100% were EGFP positive ( Fig.13 C). Therefore, it is believed that if testicular cells are microinsemination (ICSI / ROSI) is performed, the possibility of obtaining offspring is higher. Therefore, microinsemination (ICSI / ROSI) was performed using testicular cells of chimeric mice. Figure 4 , step 10).

[0323] As a result, MHC humanized ES cells were successfully propagated and MHC humanized mice that emit green fluorescence throughout the body were obtained ( Fig.14 A, B). In addition, genomic PCR also confirmed that EGFP-positive individuals retained the sequence of the human MHC region ( Fig.14 C). From these results, it can be seen that MHC humanized (xenogeneic) mice were successfully established.

[0324] Preparation of MHC humanized mice based on natural mating of chimeric mice

[0325] The results so far show that the reproductive capacity of MHC humanized chimeric mice prepared from B6-derived ES cells is low. Therefore, MHC humanization of XO ES cells (B6 / CBA F1) produced as females was also performed to prepare chimeric mice ( Fig.15 A)( Figure 4 , step 9), and mated with wild-type mice.

[0326] As a result, MHC humanized heterologous mice were successfully obtained through natural mating ( Fig.15 BD)( Figure 4 , step 10).

[0327] 4. Analysis of human MHC gene expression in MHC humanized (xenogeneic) mice

[0328] <Method>

[0329] Expression analysis of human MHC (also known as HLA) proteins

[0330] In the preparation of spleen cells, the removed spleen is first minced with a knife, and the frosted part of the slide is used to grind the tissue slices. Next, the cells are hemolyzed with 0.83% ammonium chloride, and spleen cells are prepared through a nylon mesh. Cells were stained by flow cytometry using Pacific Blue labeled anti-mouse CD 19 antibody (Biolegend), APC Conjugation Kit-Lightning-Link (Abcam) and AP C-labeled anti-HLA class I antibody (MBL) or APC-labeled anti-HLA-DR, DP, DQ antibody (Biolegend), and analyzed using FACS AriaIII (BD). In the analysis based on Western blotting, detection was performed using anti-HLA-A, B, C antibodies (MBL), anti-HLA-A antibodies (Abcam), anti-HLA-DRA antibodies (Abcam) or HRP-labeled anti-ACTB antibodies (Genscript).

[0331] In the preparation of lung-derived fibroblasts, the excised lung tissue was first minced with a knife and treated with 0.1% collagenase A (Roche) for 1 hour, the cells were freed with a pipette, and then passed through a nylon mesh to prepare lung-derived fibroblasts. Lung-derived fibroblasts were cultured in DMEM supplemented with 10% FCS.

[0332] result

[0333] The expression of human MHC genes in spleen cells was analyzed at the protein level. First, spleen cells were prepared from MHC humanized (heterogeneous) mice (Het) established from wild-type (Wt) and XO ES cells, and the expression of human MHC proteins was analyzed by Western blotting using anti-HLA-A, B, C antibodies, anti-HLA-A antibodies, and anti-HLA-DRA antibodies. As a result, the expression of human MHC in Het-derived spleen cells was specifically detected ( Fig.16 A).

[0334] Next, the expression of HLA proteins on the cell surface was analyzed. Splenocytes are mainly composed of B cells and T cells, and it should be confirmed that MHC class I is expressed in all cells and MHC class II is expressed in B cells. Therefore, when using antibodies against CD19, which is a B cell marker, anti-HLA class I antibodies that recognize human MHC class I, and anti-HLA-DP, DQ, and DR antibodies that recognize human MHC class II and analyzing them by flow cytometry, it was confirmed that HLA class I was detected in all Het-derived spleen cells, and HLA-DP, DQ, and DR were specifically expressed in Het-derived B cells ( Fig.16 B).

[0335] Sequence Listing Free Text

[0336] SEQ ID NO.1: Synthetic DNA

[0337] Additional Information:

[0338] / label="EF1" (location: 30..1217)

[0339] / label="Hyg" (location: 1230..2267)

[0340] / label="RGpA" (location: 2274..2808)

[0341] complement / label = "Origin pMB1 ori (pUC)" (location: 3562..4176)

[0342] complement / label = "Bacterial Selection Amp" (located at: 4336..5196)

[0343] SEQ ID NO.2: Synthetic DNA

[0344] Additional Information:

[0345] / label="FRT" (location: 18..65)

[0346] complement / label = "BGHpA" (location: 66..342)

[0347] complement / label = "3'Neo" (location: 347..805)

[0348] / label = "SA" (existing location: 806..871)

[0349] complement / label = "loxP" (existing position: 872..905)

[0350] SEQ ID NO.3~8: Synthetic DNA

[0351] SEQ ID NO.9: Synthetic DNA

[0352] Additional Information:

[0353] / lebel="P2A" / note="P2A" (location: 46..111)

[0354] / lebel="mRuby2" (location: 118..831)

[0355] SEQ ID NO.10~11: Synthetic DNA

[0356] SEQ ID NO.12: Synthetic DNA

[0357] Additional Information:

[0358] / label="EF1" (location: 30..1217)

[0359] / label="EGFP" (location: 1230..1949)

[0360] / label="RGpA" (location: 1956..2490)

[0361] complement / label = "Origin pMB1 ori (pUC)" (location: 3244..3858)

[0362] / label = "Bacterial Selection Amp" (location: 4018..4878)

[0363] SEQ ID NO.13: Synthetic DNA

[0364] Additional Information:

[0365] complement / label = "Puro" (location: 22..618)

[0366] complement / label = "T2A" (existing position: 619..681)

[0367] / label = "SA" (location: 684..750)

[0368] complement / label = "loxP" (existing position: 751..784)

[0369] / label = "SD" (location: 788..854)

[0370] complement / label = "5'Neo" (existing position: 855..1199)

[0371] complement / label = "P2A" (existing position: 1200..1265)

[0372] SEQ ID NO.14~21: Synthetic DNA

[0373] SEQ ID NO.22: Synthetic DNA

[0374] Additional Information:

[0375] / label="Amp" (location: 819..1679)

[0376] / label="Origin pMB1 ori(pUC)" (location: 1839..2453)

[0377] / label="LA" (location: 2957..4041)

[0378] / label="CAG promoter" (location: 4047..5746)

[0379] / label="Hprt" (location: 5756..6412)

[0380] / label="RGpA" (location: 6439..6968)

[0381] / label = "RA" (location: 6975..8363)

[0382] SEQ ID NO.23: Synthetic DNA

[0383] Additional Information:

[0384] / label="Blasticidin resistance gene" (Existence location: 126..524)

[0385] SEQ ID NO.24: Synthetic DNA

[0386] Additional Information:

[0387] / label="U6 promoter" (location: 1..249)

[0388] / label = "chimeric guide RNA scaffold" (location: 268..343)

[0389] / label="U6 terminator" (location: 344..349)

[0390] / label="CBh" (location: 440..1238)

[0391] / label="3xFLAG" (location: 1251..1319)

[0392] / label="NLS(1)" (location: 1320..1370)

[0393] / label="hSpCsn1" (location: 1371..5471)

[0394] / label="NLS" (location: 5472..5519)

[0395] / label="T2A" (location: 5526..5588)

[0396] / label="Ametrine" (location: 5589..6305)

[0397] / label="BGHpA" (location: 6315..6546)

[0398] / label = "Bacterial Selection Amp" (location: 7612..8472)

[0399] / label="Origin pMB1 ori(pUC)" (location: 8632..9246)

[0400] SEQ ID NO.27: Synthetic DNA

[0401] Additional Information:

[0402] / label="U6 promoter" (location: 1..249)

[0403] / label="chimeric guide RNA scaffold" (location: 268..353) / label="U6terminator" (location: 354..359)

[0404] / label="CBh" (location: 450..1248)

[0405] / label="3xFLAG" (location: 1261..1329)

[0406] / label="NLS(1)" (location: 1330..1380)

[0407] / label="hSpCsn1" (location: 1381..5481)

[0408] / label = "NLS" (location: 5482..5529)

[0409] / label="T2A" (location: 5536..5598)

[0410] / label="Ametrine" (location: 5599..6315)

[0411] / label="BGHpA" (location: 6325..6556)

[0412] / label = "Bacterial Selection Amp" (location: 7622..8482)

[0413] / label="Origine pMB1 ori(pUC)" (location: 8642..9256)

[0414] SEQ ID NO.28~52: Synthetic DNA

Claims

1. A vector set comprising a combination of the following targeting vectors (A), (B) and (D) or a combination of the following targeting vectors (C) and (D): (A) A targeting vector 1 is introduced into an arbitrary position P1 on the distal side of the major histocompatibility complex (human MHC) region present on the short arm of human chromosome 6, The targeting vector 1 causes translocation recombination between the region from the P1 to the distal end and the region from the position p1 corresponding to the P1 to the distal end, and the targeting vector 1 comprises a gene cassette 1, the position p1 is present on the distal side of the MHC (non-human MHC) region of the chromosome in a non-human mammal, and the gene cassette 1 comprises a recombinase recognition sequence, a partial sequence of a drug resistance gene 1 for drug screening reconstructed during translocation recombination, and a drug resistance gene 2 for drug screening after being targeted; (B) a targeting vector 2, which is introduced into a chromosome of a non-human mammal having an MHC region, into a position p1 corresponding to the P1 on the distal side of the MHC (non-human MHC) region, The targeting vector 2 causes translocation recombination between the region from the p1 to the distal end and the region from the P1 to the distal end in human chromosome 6, and the targeting vector 2 comprises a gene cassette 2, wherein the gene cassette 2 comprises a recombinase recognition sequence, other sequences of the drug resistance gene 1, and a drug resistance gene 3 for drug screening after targeting; (C) a targeting vector 3 that causes translocation recombination between a region from an arbitrary position P1 to the distal end of the distal side of the human MHC region in human chromosome 6, which is a target for cutting by a genome editing tool, and a region from a position p1 to the distal end of the distal side of the non-human MHC region in a chromosome of a non-human mammal having MHC, which corresponds to the position P1, The targeting vector 3 comprises a sequence identical to the sequence of a portion of the region R1a between the P1 and the human MHC region, a drug resistance gene 4 for drug screening after being targeted, and a sequence identical to the sequence of a portion of the region R2b on the distal side of the P1, or comprises a sequence identical to the sequence of a portion of the region R1b on the distal side of the P1, a drug resistance gene 4 for drug screening after being targeted, and a sequence identical to the sequence of a portion of the region R2a between the P1 and the non-human MHC region; (D) a targeting vector 4 that causes translocation recombination between a region from an arbitrary position P2 on the proximal side of the human MHC region that is a target for cleavage by a genome editing tool to the distal end and a region from a position p2 on the proximal side of the non-human MHC region corresponding to P2 to the distal end in a chromosome of a non-human mammal having non-human MHC, The targeting vector 4 contains the same sequence as the sequence of a portion of the region R3a on the proximal side of the P2, a drug resistance gene 5 for drug screening after targeting, and the same sequence as the sequence of a portion of the region R4b between the P2 and the non-human MHC region, or contains the same sequence as the sequence of a portion of the region R3b between the P2 and the human MHC region, a drug resistance gene 5 for drug screening after targeting, and the same sequence as the sequence of a portion of the region R4a on the proximal side of the P2.

2. The vector set according to claim 1, further comprising the following vectors (E) and (F): (E) a genome editing tool vector 1, which cuts human chromosome 6 at the arbitrary position P1; (F) A genome editing tool vector 2, which cuts the non-human chromosome at the arbitrary position p1.

3. The vector set according to claim 1, further comprising the following vectors (G) and (H): (G) a genome editing tool vector 3, which cuts human chromosome 6 at the arbitrary position P2; (H) A genome editing tool vector 4, which cuts the non-human chromosome at the arbitrary position p2.

4. The vector set according to any one of claims 1 to 3, wherein The human MHC region includes a class I region, a class II region, a class III region, or a combination thereof.

5. The carrier set according to claim 4, wherein: The class I region comprises at least one selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, MICA, and MICB.

6. The carrier set according to claim 4, wherein: The class II region includes at least one selected from HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO.

7. The vector set according to claim 4, wherein: A region containing a class I region is a region containing HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, MICA, and MICB.

8. The vector set according to claim 4, wherein The region containing the class II region is a region containing HLA-DP, HLA-DQ, HLA-DR, HLA-DM, and HLA-DO.

9. The carrier set according to claim 4, wherein: The region including both the class I region and the class II region is the region between the DAXX locus and the MOG locus.

10. The vector set according to any one of claims 1 to 3, wherein The arbitrary position P1 is the position between the MOG locus and the GABBR1 locus.

11. The vector set according to any one of claims 1 to 3, wherein The arbitrary position P2 is the position between the KIFC1 locus and the DAXX locus.

12. The vector set according to any one of claims 1 to 3, wherein Non-human mammals are rodents.

13. The vector set according to claim 12, wherein: The rodent is a mouse.

14. The vector set according to claim 13, wherein: The arbitrary position p1 is the position between the Mog locus and the Gabbr1 locus.

15. The vector set according to claim 13, wherein The arbitrary position p2 is the position between the Kifc1 locus and the Daxx locus.

16. The vector set according to any one of claims 1 to 3, wherein Drug resistance gene 1 is a neomycin resistance gene, drug resistance gene 2 is a hygromycin resistance gene and / or drug resistance gene 3 is a puromycin resistance gene.

17. The vector set according to any one of claims 1 to 3, wherein The drug resistance gene 4 is a neomycin resistance gene and / or the drug resistance gene 5 is a blasticidin resistance gene.

18. The vector set according to any one of claims 1 to 3, wherein The recombinase recognition sequence in gene cassette 1 is loxP, drug resistance gene 1 is a neomycin resistance gene, and drug resistance gene 2 is a hygromycin resistance gene.

19. The vector set according to any one of claims 1 to 3, wherein Gene cassette 1 comprises a construct represented by the following formula (1): BGHpA-3'Neo-SA-loxP-EF1-Hyg-P2A-mRuby2-RGpA(1) In the formula, BGHpA represents the poly A tailing signal sequence derived from bovine growth hormone, 3'Neo represents the sequence of a part of the 3' end of the neomycin resistance gene, SA represents the splice acceptor sequence, loxP represents the recombinase recognition sequence, EF1 represents the promoter sequence derived from human elongation factor 1α, Hyg represents the hygromycin resistance gene, P2A represents the 2A sequence derived from porcine teschovirus, mRuby2 represents the red fluorescent gene, and RGp A represents the poly A tailing signal sequence derived from rabbit β-globin.

20. The vector set according to any one of claims 1 to 3, wherein The recombinase recognition sequence in gene cassette 2 is loxP, drug resistance gene 1 is a neomycin resistance gene, and drug resistance gene 3 is a puromycin resistance gene.

21. The vector set according to any one of claims 1 to 3, wherein Gene cassette 2 comprises a construct represented by the following formula (2): EF1-EGFP-P2A-5'Neo-SD-loxP-SA-T2A-Puro-RGpA(2) In the formula, EF1 represents the promoter sequence derived from human elongation factor 1α, EGFP represents the green fluorescent gene, P2A represents the 2A sequence derived from porcine teschovirus, 5'Neo represents the sequence of a part of the 5' end of the neomycin resistance gene, SD represents the splice donor sequence, loxP represents the recombinase recognition sequence, SA represents the splice acceptor sequence, T2A represents the 2A sequence derived from Thosea asigna virus, Puro represents the puromycin resistance gene, and RGpA represents the poly A tailing signal sequence derived from rabbit β-globin.

22. A non-human mammalian cell in which the MHC region has been humanized, comprising: A chromosome obtained by replacing the MHC region of a chromosome of a non-human mammal with the MHC region of a human chromosome using the vector set according to any one of claims 1 to 3.

23. The cell according to claim 22, which is an ES cell.

24. A non-human mammal with a humanized MHC region produced from the cell of claim 22.

25. The non-human mammal according to claim 24, wherein Non-human mammals are rodents.

26. The non-human mammal according to claim 25, wherein The rodent is a mouse.

27. The non-human mammal according to claim 26, comprising: a chromosome obtained by replacing the chromosome portion of at least the portion of ...

28. A method for producing a non-human mammal with a humanized MHC region, comprising the following steps: (1) a step of preparing a cell comprising a chromosome in which the MHC region of a non-human mammal chromosome is replaced with the MHC region of a human chromosome, using the vector set according to any one of claims 1 to 3; and (2) A step of obtaining a non-human mammal whose MHC region has been humanized from the cells obtained in (1).

29. The method according to claim 28, wherein: Non-human mammals are rodents.

30. The method of claim 29, wherein: The rodent is a mouse.

31. A method for producing a non-human mammal having a humanized MHC region, comprising: A process for producing a non-human mammal having a chromosome in which the MH C region has been humanized by transplanting the cell according to claim 22 into a pseudo-parent of a non-human mammal.

32. The method according to claim 31, wherein: Non-human mammals are rodents.

33. The method of claim 32, wherein: The rodent is a mouse.

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