HK1178206B - Mouse artificial chromosome vector - Google Patents

Abstract

A naturally occurring centromere derived from a mouse chromosome; a long arm fragment derived from a mouse chromosome, which is produced by deleting a distal long arm from a mouse chromosome long arm which is located adjacent to centromere; a mouse artificial chromosome vector characterized by containing a telomere sequence and capable of being retained stably in a mammalian cell and an individual tissue; a cell or a non-human animal which carries the vector; and use of the cell or the non-human animal.

Description

Mouse artificial chromosome vector

Technical Field

The present invention relates to a mouse artificial chromosome vector which is stably carried in rodent and can make progeny transmission (daughter YunDa).

The invention also relates to a cell carrying the mouse artificial chromosome vector.

The present invention further relates to a non-human animal such as a mouse carrying the mouse artificial chromosome vector.

Background

Transgenic mice are widely used because they utilize target genes and expression products by introducing gene-carrying vectors. However, in the transgenic mice so far, the sites of gene introduction are random, and the expression of the introduced gene is sometimes suppressed by the site effect of the introduction, and the copy number cannot be controlled in the conventional gene introduction method, and the size is limited to about 200 kb. Thus, it is difficult to clone genes or gene clusters of sizes exceeding 200kb, which are not uncommon for mammalian genes, onto vectors containing control regions. Therefore, the conventional gene transfer method has a limitation that the original function of the transferred gene cannot be reproduced or examined.

In order to solve such a problem, the present inventors have developed a technique for preparing a chromosome-introduced mouse using a novel chromosome introduction method in which a gene is introduced at the chromosome level (non-patent document 1). As a result of producing a chimeric mouse by introducing a human chromosome or a fragment thereof into a mouse Embryonic Stem (ES) cell by this technique, it was revealed that human chromosome fragments are carried independently, a plurality of human genes are expressed with tissue specificity, and there are also human chromosomes which can be transmitted as progeny by meiosis. The present inventors introduced the total length of human chromosome 21 (about 35Mb) into a mouse and produced a down syndrome model mouse that is also highly valuable in practical terms (non-patent document 2). The mouse was analyzed, and as a result, the gene introduced into chromosome 21 of human reproduced a physiological expression pattern, indicating the effectiveness of the chromosomal vector.

Furthermore, the present inventors have also shown that a Human Artificial Chromosome (HAC) vector containing a megabase (Mb) sized specific Human chromosome region can be successfully constructed and functions in mouse individuals by a chromosome engineering technique, that is, by chromosome deletion using the telomere truncation technique of an Artificial telomere sequence and chromosome cloning using the Cre/loxP system, thereby constructing a Human Artificial chromosome containing only the target region(s) (non-patent document 3). Further, a novel HAC vector containing no known gene was constructed by applying the above-mentioned technique (non-patent document 4). The present inventors have also succeeded in stably expressing a target gene by introducing a HAC vector carrying the target gene into any cell based on the above-mentioned background. Further, as an example of modeling a human mouse using the HAC vector, a drug metabolizing enzyme CYP3A gene cluster (1Mb) on human chromosome 7 and a human DMD gene (2.5Mb) which is a causative gene of human X-linked muscular dystrophy were cloned into HAC vectors (CYP3A-HAC and DMD-HAC), and introduced into mouse ES cells to prepare a mouse (patent document 1, non-patent document 5).

Analysis of the tissue carrying rate and expression of mice into which CYP3A-HAC has been introduced revealed that the CYP3A gene cluster on HAC is carried in each tissue of mice (FIG. 8 of patent document 1), and its expression pattern is the same as that in human tissues, and it is specifically expressed in the liver and small intestine. Further, analysis of the tissue carrying rate and expression analysis of the introduction of DMD-HAC into mice revealed that DMD-HAC is carried in each tissue of mice (fig. 4A of non-patent document 5), and it is known that at least 3 of splice isoforms expressed specifically in human tissues are expressed in the same manner as in human. These results suggest the usefulness of the introduced HAC mouse as a substitute for the conventional transgenic mouse for a new gene (group) introduction method.

Mammalian artificial chromosome vectors containing human artificial chromosomes have advantages that are not found in conventional vector systems (viruses, YACs, BACs, PACs, cosmids, and plasmids), and therefore, are expected to be used as a system for functional analysis of new genes and for creating human model animals. For example, patent documents 2 and 3 disclose HAC vectors that are composed of fragments of chromosomes obtained by modifying and reducing the size of human chromosome 14 or human chromosome 21 and that are relatively stably carried in cells.

However, there are problems in that human chromosome 21-introduced mice (down syndrome model mice) or HAC vector-introduced mice, which make it possible to introduce Mb units that were not possible in conventional genetically modified mice, have the following: the human chromosome vectors have reduced carriage rates, inter-organizational and inter-individual variation, and unstable frequency of progeny transmission. Therefore, it is often necessary to consider the carrying rate of HAC vectors and the like, and in some cases, it is difficult to accurately analyze the expression dynamics and expression products of target genes in detail at the tissue cell level in the case of studying the functions of specific gene regions and the relationship with diseases, and thus homogeneous analysis with high reproducibility is hindered.

Furthermore, when mouse cells and human cells are subjected to cell fusion, it is known that human chromosomes are unstable in mouse cells. In this way, since the human chromosome containing the human artificial chromosome vector is not carried in a constant rate in mouse cells, the advantages as an artificial chromosome vector cannot be fully exerted in cases where the human artificial chromosome vector is introduced into mouse cells or in cases where transgenic mice are prepared. In the future, by increasing the carrying rate of an introduced gene or making it constant on the basis of the production of a transgenic mouse cell or a transgenic mouse, more detailed and accurate analysis of a gene function with high reproducibility or effective recovery of a gene expression product can be performed.

Documents of the prior art

Patent document

Patent document 1 international publication WO2009/063722 pamphlet

WO2004/031385 on International publication No. WO2 of patent document 2

Patent document 3 Japanese laid-open patent application No. 2007-295860

Non-patent document

Non-patent document 1Tomizuka et al, Nat Genet, 16: 133-143, 1997

Non-patent document 2Shinohara et al, HMG, 10: 1163-75, 2001

Non-patent document 3Kuroiwa et al, Nat Biotech, 18: 1086-1090, 2000

Non-patent document 4Katoh et al, BBRC, 321: 280-290, 2000

Non-patent document 5Hoshiya et al, Mol Ther, 17: 309-17, 2009

Disclosure of Invention

Problems to be solved by the invention

Many of the mammalian artificial chromosomes reported so far are human artificial chromosomes (Japanese patent laid-open No. 2005-230020; Japanese patent laid-open No. 2008-54501; Japanese patent laid-open No. 2007-306928; Japanese patent laid-open No. 2007-295860), but there are also very few reports on mouse artificial chromosomes which are characterized by utilizing the sequence of a part of mouse centromere (S.Stewart et al (2002) Gene Therapy 9: 719-723).

As described above, the human Chromosome fragment is unstable in mouse cells when the natural human Chromosome fragment is transferred into the mouse cells (e.g., Shinohara et al (2000) Chromosome Research, 8: 713-725). This also applies to individual mice, where the human artificial chromosome is unstable in each tissue of the mouse, the proportion (carrying rate) of the human artificial chromosome carried in the tissue cells of the mouse tends to decrease, and the carrying rate of the tissue cells of the mouse is not constant. Alternatively, the same applies to mouse individuals, and the carrying rate of human artificial chromosomes is not constant among mouse individuals. Due to such uneven carrying rate between mouse tissues and between individuals, it is difficult to analyze an introduced gene in detail, accurately and with high reproducibility using mouse cells into which a target gene (group) is introduced via a human artificial chromosome or mouse individuals.

As described above, with respect to artificial chromosomes derived from rodents including mice, there are very few reports related thereto, and there is no artificial chromosome that can be stably maintained in rodent cells or in an individual. The present invention aims to provide a mouse artificial chromosome vector in which an introduced target gene(s) can be stably maintained in rodent cells or rodent individuals, thereby enabling detailed, accurate and highly reproducible analysis.

Means for solving the problems

In summary, the present invention includes the following features.

(1) A mouse artificial chromosome vector which comprises a natural centromere derived from a mouse chromosome, a long-arm fragment derived from a mouse chromosome, which is obtained by deleting the distal end of the long arm from the long-arm part of a mouse chromosome in the vicinity of the centromere, and a telomere sequence, and which can be stably carried in cells and tissues of a mammal.

(2) The mouse artificial chromosome vector according to the above (1), wherein the mouse chromosome is any one of chromosomes 1to 19.

(3) The mouse artificial chromosome vector according to the above (1) or (2), wherein the long-arm fragment derived from a mouse chromosome is composed of a remaining part in which at least 99.5% of the total endogenous gene thereof is deleted from the long arm of any one of mouse chromosomes 1to 19.

(4) The mouse artificial chromosome vector according to any one of the above (1) to (3), which comprises the mouse artificial chromosome contained in the deposited cell line DT40B6bT-1(FERM BP-11128) as a basic structure.

(5) The mouse artificial chromosome vector according to any one of the above (1) to (4), wherein the mammal is a rodent.

(6) The mouse artificial chromosome vector according to the above (5), wherein the rodent is a mouse, a rat or a hamster.

(7) The mouse artificial chromosome vector according to any one of (1) to (6) above, further comprising one or more DNA sequence insertion sites.

(8) The mouse artificial chromosome vector according to the above (7), wherein the DNA sequence insertion site is a recognition site of a site-specific recombinase.

(9) The mouse artificial chromosome vector according to the above (7) or (8), wherein the DNA sequence insertion site is a loxP sequence, FRT sequence, a DNA fragment encoding the DNA fragment, and a DNA fragment encoding the DNA fragment,Andsequences, R4attB and R4attP sequences, TP901-1attB and TP901-1attP sequences, or Bxb1attB and Bxb1attP sequences.

(10) The mouse artificial chromosome vector according to any one of the above (1) to (9), further comprising a reporter gene, a selection marker gene, or both.

(11) The mouse artificial chromosome vector according to any one of (1) to (10) above, further comprising a foreign DNA sequence.

(12) The mouse artificial chromosome vector according to any one of (1) to (11) above, wherein the size of the foreign DNA sequence is 200kb or more.

(13) The mouse artificial chromosome vector according to the above (11) or (12), wherein the foreign DNA sequence is a human DNA sequence.

(14) The mouse artificial chromosome vector according to any one of the above (11) to (13), wherein the foreign DNA sequence is a DNA sequence of a gene related to drug metabolism.

(15) The mouse artificial chromosome vector according to the above (14), wherein the drug metabolism-related gene is a gene encoding an enzyme involved in the first phase reaction or the second phase reaction.

(16) The mouse artificial chromosome vector according to (15) above, wherein the enzyme involved in the first phase reaction is an enzyme encoding at least one selected from the group consisting of CYP and CES, such as CYP1A, CYP1B, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2J, CYP3A, CYP4A, CYP4B, and a subfamily thereof.

(17) The mouse artificial chromosome vector according to the above (15), wherein the enzyme involved in the second phase reaction is an enzyme encoding at least one selected from the group consisting of UGT1 and UGT 2.

(18) The mouse artificial chromosome vector according to (14) above, wherein the drug metabolism-related gene is a gene encoding a transporter.

(19) The mouse artificial chromosome vector according to the above (18), wherein the gene encoding the transporter is a gene encoding at least one selected from the group consisting of MDR1, MDR2, MRP2, OAT, OATP, OCT, and BCRP.

(20) The mouse artificial chromosome vector according to (14) above, wherein the drug metabolism-related gene is a gene encoding a nuclear receptor.

(21) The mouse artificial chromosome vector according to (20) above, wherein the gene encoding a nuclear receptor is a gene encoding at least one selected from the group consisting of PXR, AhR, CAR, and PPAR α.

(22) The mouse artificial chromosome vector according to any one of the above (11) to (13), wherein the foreign DNA sequence is a DNA sequence of a long arm or a short arm of the human chromosome.

(23) The mouse artificial chromosome vector according to any one of the above (11) to (21), wherein the foreign DNA sequence contains at least two gene sequences selected from the group consisting of a gene encoding an enzyme involved in the first phase reaction, a gene encoding an enzyme involved in the second phase reaction, a gene encoding a transporter, and a gene encoding a nuclear receptor.

(24) The mouse artificial chromosome vector according to (22) above, wherein the human chromosome is a DNA sequence of a long arm or a short arm of the human chromosome that includes a disease-causing region (the main region of the cause of the disorder Yuncheng nucleus).

(25) The mouse artificial chromosome vector according to any one of the above (11) to (13), wherein the foreign DNA sequence is a sequence of a gene or DNA encoding a polypeptide such as a cytokine, a hormone, a growth factor, a trophic factor, a hematopoietic factor, a coagulation/hemolysin factor, an immunoglobulin, a G protein-coupled receptor, or an enzyme, or a gene or DNA for therapy of a disease such as a tumor, muscular dystrophy, hemophilia, a neurodegenerative disease, an autoimmune disease, an allergic disease, or a genetic disease.

(26) The mouse artificial chromosome vector according to any one of (1) to (25) above, wherein the cell is a hepatocyte, an intestinal cell, a renal cell, a splenocyte, a lung cell, a cardiac cell, a skeletal muscle cell, a brain cell, a bone marrow cell, a lymphocyte, a megakaryocyte, a sperm, or an ovum.

(27) The mouse artificial chromosome vector according to any one of the above (1) to (25), wherein the tissue is a tissue derived from a liver, intestine, kidney, spleen, lung, heart, skeletal muscle, brain, bone marrow, testis, or ovary.

(28) A cell carrying the mouse artificial chromosome vector of any one of (1) to (27) above.

(29) The cell according to the above (28), wherein the cell is selected from the group consisting of a somatic cell, a non-human germ cell, a stem cell and a precursor cell.

(30) The cell according to the above (29), wherein the stem cell is an Embryonic Stem (ES) cell or an Induced Pluripotent Stem (iPS) cell.

(31) The cell according to any one of the above (28) to (30), wherein the cell is a primary culture cell, a secondary cell or a cell line.

(32) The cell according to any one of (28) to (31) above, wherein the cell is a cell that can produce a human antibody.

(33) A pharmaceutical composition comprising the cell according to any one of (28) to (32) above carrying a mouse artificial chromosome vector containing a foreign DNA sequence for treating a disease.

(34) A non-human animal carrying the mouse artificial chromosome vector of any one of (1) to (27) above.

(35) The non-human animal according to (34) above, which is a disease model animal.

(36) The non-human animal according to (34) above, which is an animal capable of expressing a foreign gene related to human drug metabolism.

(37) The non-human animal according to (34) above, which is an animal capable of producing a human antibody.

(38) The non-human animal according to any one of (34) to (37) above, wherein an endogenous gene corresponding to the exogenous DNA contained in the mouse artificial chromosome vector is disrupted or the expression of the endogenous gene is reduced.

(39) A method for producing a protein, which comprises culturing a cell of any one of (28) to (32) above carrying a mouse artificial chromosome vector containing a foreign DNA sequence and recovering a protein encoded by the DNA produced.

(40) A method for producing a human antibody, which comprises producing a human antibody using the non-human animal according to (37) or (38) above which carries a mouse artificial chromosome vector containing a human antibody gene, and recovering the antibody.

(41) A method for screening a substance effective for treating a disease, which comprises administering a candidate drug to the non-human animal according to (35) above as a disease model animal, and evaluating the therapeutic effect of the drug.

(42) A method for testing pharmacological action and/or metabolism and/or toxicity of a drug or food, which comprises administering the drug or food to the non-human animal as described in (36) or (38) above or to a cell, organ or tissue derived from the non-human animal carrying a mouse artificial chromosome vector containing a gene related to human drug metabolism, and measuring the pharmacological action and/or metabolism and/or toxicity of the drug or food.

(43) A method for testing toxicity of a drug or food, which comprises culturing microsome or microsome fraction S9 obtained from the non-human animal of (36) or (38) above, which carries a mouse artificial chromosome vector containing a gene associated with metabolism of a human drug, together with cultured cells or bacteria and a drug and/or food, and measuring the effect of the drug or food on the cells or bacteria.

(44) A method for stabilizing a large-sized DNA in a cell or an individual, which comprises using the mouse artificial chromosome vector of any one of (1) to (27) above, and stably maintaining a large-sized foreign DNA of 200kb or more in a rodent cell or a rodent individual at a carrying rate of 90% or more.

According to the present invention, when a target gene (set) is introduced into a rodent cell or a rodent individual, a mouse artificial chromosome vector provided with a DNA sequence insertion site can stably and uniformly carry the target gene (set) in all cells or tissues that have been difficult in the past, and can co-insert a reporter gene with a target foreign DNA sequence or gene, so that the introduction of the vector into the cell can be visualized, and analysis in detail and with high accuracy and reproducibility and effective recovery of an expression product can be performed.

The present specification includes the contents described in the specification and/or drawings of japanese patent application No. 2010-1425, which is the priority base of the present application.

Drawings

FIG. 1 shows a schematic diagram of the steps of examples 1 and 2. The cell names and the like in the figure are expressed as follows. Cell name (intracellular gene engineering; carrying chromosome fragment name, import-carrying chromosome name). The symbols and the like in the drawings are as follows. BSr: blasticidin (BS) resistance gene; puro: a puromycin resistance gene; artificial telomeres: an artificial telomere (TTAGGG) repeat; EGFP: a green fluorescent protein expression gene; neo: a neomycin (G418) resistance gene; loxP: a site-specific DNA sequence insertion site; 3' HPRT: the sequence of exons 3 to 9 of the HPRT gene;

FIG. 2 shows a schematic representation of the steps of example 3. The cell names and the like in the figure are expressed as follows. Cell name (intracellular gene engineering; carrying chromosome fragment name, import-carrying chromosome name). The symbols and the like in the drawings are as follows. hChr 7: human chromosome 7; CYP3 Acluster: the human CYP3A gene cluster; 5' HPRT: the sequences of exons No. 1 and No. 2 of the HPRT gene; loxP: a site-specific DNA sequence insertion site; hyg: a hygromycin resistance gene; and (h) hisD: a histidinol resistance gene; artificial telomeres: an artificial telomere (TTAGGG) repeat; EGFP: a green fluorescent protein expression gene; neo: a neomycin (G418) resistance gene; 3' HPRT: the sequence of exons 3 to 9 of the HPRT gene; puro: a puromycin resistance gene;

FIG. 3 shows a schematic representation of the steps of example 4. The cell names and the like in the figure are expressed as follows. Cell name (intracellular gene engineering; carrying chromosome fragment name, import-carrying chromosome name). The symbols and the like in the drawings are as follows. puro: a puromycin resistance gene; artificial telomeres: an artificial telomere (TTAGGG) repeat; 5' HPRT: the sequences of exons No. 1 and No. 2 of the HPRT gene; hyg: a hygromycin resistance gene; loxP: a site-specific DNA sequence insertion site;

FIG. 4 shows a schematic representation of the steps of example 5. The cell names and the like in the figure are expressed as follows. Cell name (intracellular gene engineering; carrying chromosome fragment name, import-carrying chromosome name). The symbols and the like in the drawings are as follows. puro: a puromycin resistance gene; artificial telomeres: an artificial telomere (TTAGGG) repeat; neo: a neomycin (G418) resistance gene; loxP: a site-specific DNA sequence insertion site; 3' HPRT: the sequence of exons 3 to 9 of the HPRT gene;

FIG. 5 shows a schematic representation of the steps of example 6. The cell names and the like in the figure are expressed as follows. Cell name (intracellular gene engineering; carrying chromosome fragment name, import-carrying chromosome name). The symbols and the like in the drawings are as follows. neo: a neomycin (G418) resistance gene; loxP: a site-specific DNA sequence insertion site; 3' HPRT: the sequence of exons 3 to 9 of the HPRT gene; puro: a puromycin resistance gene; artificial telomeres: an artificial telomere (TTAGGG) repeat; EGFP: a green fluorescent protein expression gene; 5' HPRT: the sequences of exons No. 1 and No. 2 of the HPRT gene;

FIG. 6 shows cell fusion clones of mouse A9 cells (mouse A9(neo)) (left) and mouse A9 cells and mouse fibroblasts (mouse embryonic fibroblasts (mCHR11-BSr)) (mouse A9x mouse embryonic fibroblast hybrid (neo; mCHR 11-BSr);

FIG. 7 shows the results of FISH analysis of DT40(mCHr11-Bsr) clones using mouse Cot-1DNA as a probe.

FIG. 8 shows the results of SKY FISH analysis (left) and SKY FISH chromatin image (right) of mouse chromosome 11 (mCHr11-BSr) introduced into chicken DT40 cells;

FIG. 9 shows a partial structure of a vector for telomere truncation in the AL671968 region of mouse chromosome 11 and an allele of mouse chromosome 11 that is homologously recombined using the vector;

FIG. 10 shows the results of monochromatic FISH analysis of DT40(MAC) [ DT40(B6bT-1) ] clones containing alleles of mouse artificial chromosome MAC, telomere truncated in mouse chromosome 11 region AL671968 using the pBS-TEL/puro MAC vector (right). DT40(mCHr11-BSr) in the left panel represents DT40(mCHr11-BSr) clones before telomere truncation was performed;

FIG. 11 shows partial structures of alleles of a GFP-neo-loxP-3' HPRT type loxP targeting vector (pMAC1) and mouse artificial chromosome MAC using the vector for homologous recombination;

FIG. 12 shows the results of two-color FISH analysis of DT40(MAC1) clones using mouse Cot-1DNA and GFP-PGKneo-loxP-3' HPRT cassette as probes;

FIG. 13 shows the results of a monochromatic FISH analysis of CHO (HPRT-; MAC1) clones using mouse Cot-1DNA as a probe;

FIG. 14 shows the results of monochromatic FISH analysis of mouse ES (MAC1) clones using mouse minor satellite (minor satellite) DNA as a probe;

FIG. 15 shows a targeting vector (pMPloxPhyg) for inserting loxP into the AC004922 region located near the CYP3A locus on human chromosome 7 and on the centromeric side (about 300Kb on the centromeric side), and the partial structure of human chromosome 7 alleles which are homologously recombined using this vector (FIG. 15 a). FIG. 15b shows the results of DNA hybridization analysis of homologous recombinants in hygromycin-resistant strains cloned from DT40 cells containing the human chromosome 7 fragment after transfection with the linearized vector. For the arrows in FIG. 15b, the top arrow indicates non-homologous recombinants (about 10.9kb), and the bottom arrow indicates homologous recombinants (about 8.9 kb);

FIG. 16 shows the partial structure of a targeting vector (pTELhisD-PT) for inserting human telomere sequences into the AC073842 region located near the CYP3A locus on human chromosome 7 and telomeric (about 150Kb telomere) and human chromosome 7 alleles using this vector for homologous recombination;

FIG. 17 shows the results of two-color FISH analysis of CHO (HPRT-, MAC1+ hCHr7-loxP-tel) clones using mouse Cot-1DNA and human Cot-1DNA as probes.

FIG. 18 shows a construction of mouse artificial chromosome CYP3A-MAC obtained by transposing and cloning about 1Mb of a region around the human CYP3A gene cluster region (AC 004922-human CYP3A gene cluster-AC 073842) into MAC 1;

FIG. 19 shows the results of two-color FISH analysis of CHO (CYP3A-MAC, hCHr 7-. DELTA.CYP 3A) clones using human Cot-1DNA and mouse Cot-1DNA as probes;

FIG. 20 shows partial structures of alleles of a targeting vector (pMAC2) for constructing mouse artificial chromosome vector MAC2 and mouse artificial chromosome MAC obtained by homologous recombination using the vector;

FIG. 21 shows the results of two-color FISH analysis of DT40(MAC2) clones using mouse Cot-1DNA and 5' HPRT-loxP-PGKhygro cassette as probes;

FIG. 22 shows the results of two-color FISH analysis of CHO (HPRT-; MAC2) clones using mouse Cot-1DNA and 5' HPRT-loxP-PGKhygro cassette as probes;

FIG. 23 shows the results of monochromatic FISH analysis of mouse ES (HPRT-, MAC2) clones using mouse minor satellite DNA as a probe;

FIG. 24 shows partial structures of alleles of PGKneo-loxP-3' HPRT type loxP targeting vector (pMAC3) and mouse artificial chromosome MAC3 which is homologously recombined with the vector;

FIG. 25 shows the results of two-color FISH analysis of DT40(MAC3) clones using mouse Cot-1DNA and mouse secondary satellite DNA as probes;

FIG. 26 shows the results of a monochromatic FISH analysis of CHO (HPRT-; MAC3) clones using mouse Cot-1DNA as a probe;

FIG. 27 shows the results of monochromatic FISH analysis of the drug-resistant clone B6 (HPRT-; MAC3) using mouse secondary satellite DNA as a probe;

FIG. 28 shows the results of analysis of carrying rate of long-term culture of B6 (HPRT-; MAC3) clone. The solid line represents B6 (HPRT-: MAC3) -3, and the dotted line represents the MAC carrier carrying rate of B6 (HPRT-: MAC3) -s 6.

FIG. 29 shows a procedure for site-specific gene insertion of a specific gene (for example, GFP) into mouse artificial chromosome vector MAC3 by Cre-loxP method, and shows the partial structure of a vector for GFP insertion and mouse chromosome 11 allele by homologous recombination using the vector;

FIG. 30 shows the results of two-color FISH analysis of CHO (GFP-MAC) clones carrying mouse artificial chromosome GFP-MAC using mouse Cot-1DNA and X6.1EGFP as probes;

FIG. 31 shows the results of FISH analysis after long-term culture of mouse artificial chromosome GFP-MAC in mouse ES cells (B6-ES strain) using mouse secondary satellite DNA and GFP as probes;

FIG. 32 shows the results of analysis of the carrying rate of long-term culture of B6(GFP-MAC) clone. Diamonds represent the carrying rate of long-term cultures with drug selection, squares represent the carrying rate of long-term cultures without drug selection;

FIG. 33 shows progeny transmission individuals produced by chimeric mice carrying a mouse artificial chromosome vector (GFP-MAC);

FIG. 34 shows the carrying rates of 21HAC1 or 21HAC2 and MAC1 in CHO cells after long-term culture (25 PDL);

FIG. 35 shows the carrying rate of 21HAC2 or MAC1 in ES cells after long-term culture (75 PDL);

FIG. 36 shows a solid fluorescence microscope image in TC (MAC1) mouse (female) tissue;

FIG. 37 shows the GFP positivity in blood system cells of TC (MAC1) or TC (21HAC2) mice derived from bone marrow cells;

FIG. 38 is a graph showing the GFP-positivity in blood system cells of spleen-derived cells of a TC (MAC1) mouse or a TC (21HAC2) mouse;

FIG. 39 shows the results of a monochromatic FISH analysis of tail fibroblasts from TC (MAC1) mice using mouse secondary satellite DNA probes;

FIG. 40 shows the results of a two-color FISH analysis of A9(CYP3A-MAC) using CYP3A-BAC (RP11-757A13) and a mouse secondary satellite DNA probe;

FIG. 41 shows the results of monochromatic FISH analysis of TT2F (CYP3A-MAC) using CYP3A-BAC (RP11-757A13) DNA probe;

FIG. 42 shows CYP3A-MAC carriage rate in ES cells after long-term culture (100 PDL);

FIG. 43 shows a solid fluorescence microscope image in TC (CYP3A-MAC) mouse (male) tissue;

FIG. 44 shows the GFP positivity in blood system cells of bone marrow-derived cells of TC (CYP3A-MAC) or TC (CYP3A-HAC Δ) mice;

FIG. 45 shows the carrying rates of CYP3A-MAC or CYP3A-HAC Δ in various tissues of TC (CYP3A-MAC) or TC (CYP3A-HAC Δ) mice;

FIG. 46 shows the results of monochromatic FISH analysis in TC (CYP3A-MAC) heterozygous or TC (CYP3A-MAC) homozygous mice using CYP3A-BAC (RP11-757A13) DNA probes;

FIG. 47 is a graph showing the results of tissue-specific gene expression analysis of the CYP3A gene cluster in each tissue of TC (CYP3A-MAC) mice. GAPDH represents glyceraldehyde3-phosphate dehydrogenase (glyceraldehyde 3-phosphatedehydrogenase);

FIG. 48 is a graph showing the results of a time-specific gene expression analysis of the CYP3A gene cluster in the liver of TC (CYP3A-MAC) mouse;

FIG. 49 shows the results of two-color FISH analysis of rat ES (CYP3A-MAC) using CYP3A-BAC (RP11-757A13) and mouse Cot-1DNA probes;

FIG. 50 shows the results of two-color FISH analysis of CHO (HPRT-; MAC1, hCHr21-loxP) using mouse Cot-1DNA and human Cot-1DNA probes;

FIG. 51 shows the construction of mouse artificial chromosome hCHr21q-MAC obtained by transposing about 33Mb of the hCHr21q region into MAC 1;

FIG. 52 shows the results of two-color FISH analysis of CHO (hCHr21q-MAC, hCHr21-hCHr21q) clones using human Cot-1DNA and mouse Cot-1DNA as probes;

FIG. 53 shows the results of a two-color FISH analysis of TT2F (hCHr21q-MAC) clones using human Cot-1DNA and mouse secondary satellite DNA probes;

FIG. 54 is a graph showing the hCHr21q-MAC carrying rate in ES cells after long-term culture (50 PDL);

FIG. 55 shows a fluorescent photograph of chimeric mice carrying hCHr21 q-MAC;

FIG. 56 shows a targeting vector (pCKloxyphyg) for inserting a loxP sequence into AP001721 proximal to DSCR (Down syndrome-causing region) of human chromosome 21 (hCHr21), a target sequence, and a chromosomal allele produced by homologous recombination;

FIG. 57 shows the result of southern blot analysis in DT40 (hChr21q22.12-loxP);

FIG. 58 shows the results of two-color FISH analysis of DT40(hChr21q22.12-loxP) clone using human Cot-1DNA and hygromycin DNA as probes;

FIG. 59 shows the results of two-color FISH analysis of CHO (HPRT-; MAC1, hChr21q22.12-loxP) clones using human Cot-1DNA and mouse Cot-1DNA as probes;

FIG. 60 shows the construction of mouse artificial chromosome hChr21q22.12-MAC obtained by transposing about 12Mb of the hChr21q22.12-qter region into MAC 1;

FIG. 61 shows the results of two-color FISH analysis of CHO (hCH21q22.12-MAC, hCHr 21-hCH21q22.12) clones using human Cot-1DNA and mouse Cot-1DNA as probes;

FIG. 62 shows the results of two-color FISH analysis of TT2F (hChr21q22.12-MAC) clones using human Cot-1DNA and mouse secondary satellite DNA probes;

FIG. 63 shows the carriage rate of hChr21q22.12-MAC in ES cells after long-term culture (50 PDL);

FIG. 64 shows the carrying rates of 21HAC1 or 21HAC2 and MAC2 in CHO cells after long-term culture (25 PDL);

FIG. 65 shows the structure of 1 copy FVIII-PAC;

FIG. 66 shows a method for constructing 1-16 copies of FVIII-PAC;

FIG. 67 shows the results of agglutination assays (comparison of FVIII activity) after long-term culture in CHO (FVIIIx1-MAC)1-3 and CHO (FVIIIx1-HAC) 1-2;

FIG. 68 shows the results of agglutination assays in CHO (FVIIIx1-MAC) and CHO (FVIIIx16-MAC) (comparison of FVIII activity);

FIG. 69 shows an entry vector construction method for constructing a multigene integration site (multi-integrase platform) cassette;

FIG. 70 shows a method for constructing a multiple gene-carrying site (multiple integrase platform) cassette;

FIG. 71 shows a method of constructing an MI-MAC vector;

FIG. 72 shows a method of inserting a gene into an MI-MAC vector;

FIG. 73 shows a PXR-MAC construction method;

FIG. 74 shows the results of two-color FISH analysis of CHO (PXR-MAC) clones using mouse cot-1DNA and human PXR-BAC derived DNA (RP11-169N13) (CHORI) as probes;

FIG. 75 shows the results of monochromatic FISH analysis of TT2F (PXR-MAC) clones using human PXR-BAC-derived DNA (RP11-169N13) (CHORI) as a probe;

FIG. 76 shows partial structures of alleles of GFP-5' HPRT-loxP-hyg type loxP targeting vector (pMAC4) and mouse artificial chromosome MAC4 that was homologously recombined with the vector;

FIG. 77 shows the results of two-color FISH analysis of DT40(MAC4) clone using mouse cot-1DNA and GFP-5' HPRT-loxP-hyg cassette as probes;

FIG. 78 shows the results of a monochromatic FISH analysis of CHO (HPRT-; MAC4) clones using mouse Cot-1DNA as a probe;

FIG. 79 shows the partial structure of a targeting vector (pTELpuro-UGT2) for inserting human telomere sequences in the AC1252392 region located near the UGT2 locus on human chromosome 4 and telomeric (about 150Kb telomere) and human chromosome 4 alleles using this vector for homologous recombination;

FIG. 80 results of two-color FISH analysis of DT40(hCHr4-tel) using human cot-1DNA and puromycin DNA as probes. The left panel shows DT40 before reconstruction (hCHr4), and the right panel shows DT40 after reconstruction (hCHr 4-tel);

FIG. 81 shows a targeting vector (pUGT2loxPneo) for inserting a loxP site into AC074378 of chromosome 4 of human, a target sequence, and a chromosomal allele by homologous recombination;

FIG. 82 shows the results of two-color FISH analysis of DT40(hCHr4-loxP-tel) using human cot-1DNA and neomycin DNA as probes;

FIG. 83 shows the results of two-color FISH analysis of CHO (HPRT-; MAC4, hCHr4-loxP-tel) clones using human Cot-1DNA and mouse Cot-1DNA as probes;

FIG. 84 is a drawing showing the construction of mouse artificial chromosome UGT2-MAC obtained by transposing and cloning 2Mb of human UGT2 gene cluster region (AC 074378-human UGT2 gene cluster-AC 125239) into MAC 4;

FIG. 85 shows the results of two-color FISH analysis of CHO (UGT2-MAC, hCHr 4-. DELTA.UGT 2) clones using UGT2-BAC (RP11-643N16) (CHORI) DNA and mouse Cot-1DNA as probes;

FIG. 86 shows the results of a two-color FISH analysis of A9(UGT2-MAC) clones using UGT2-BAC (RP11-643N16) (CHORI) and mouse secondary satellite DNA as probes;

FIG. 87 shows the results of monochromatic FISH analysis of TT2F (UGT2-MAC) clones using UGT2-BAC (RP11-643N16) (CHORI) DNA as a probe;

FIG. 88 is a graph showing the UGT2-MAC carrying rate in ES cells after long-term culture (75 PDL);

FIG. 89 shows the partial structure of a targeting vector (pTELpuro-CYP2C) for inserting human telomere sequence into the AL157834 region located near the CYP2C locus on human chromosome 10 and telomeric (about 150Kb telomere) and human chromosome 10 alleles using this vector for homologous recombination;

FIG. 90 shows the results of two-color FISH analysis of DT40(hCHr10-tel) using human cot-1DNA and puromycin DNA as probes. The left panel shows DT40 before reconstruction (hCHr10), and the right panel shows DT40 after reconstruction (hCHr 10-tel);

FIG. 91 shows a targeting vector (pCYP2CloxPneo) for inserting a loxP site into AL138759 of human chromosome 10, a target sequence, and a chromosomal allele by homologous recombination;

FIG. 92 shows the results of two-color FISH analysis of clones (HPRT-; MAC4, hCHr10-loxP-tel) using mouse Cot-1DNA and human Cot-1DNA as probes;

FIG. 93 shows the construction of mouse artificial chromosome CYP2C-MAC, which is obtained by transposing 380kb around the human CYP2C gene cluster region (AL 138759-human CYP2C gene cluster-AL 157834) into MAC 4;

FIG. 94 shows the results of two-color FISH analysis of CHO (CYP2C-MAC, hCHr 10-. DELTA.CYP 2C) clone using CYP2C-BAC (RP11-466J14) (CHORI) DNA and mouse Cot-1DNA as probes;

FIG. 95 shows a targeting vector (pMDR11oxPbs) for inserting a loxP sequence into AC005045 of human chromosome 7, a target sequence, and a chromosomal allele produced by homologous recombination.

FIG. 96 shows the partial structure of a targeting vector (pTELpuro-MDR1) for inserting human telomere sequences in the AC003083 region located near the MDR1 locus and telomeric (about 50Kb telomere) on human chromosome 7 and human chromosome 7 alleles utilizing this vector for homologous recombination;

FIG. 97 shows the results of two-color FISH analysis of DT40(hCHr7M-loxP-tel) using human cot-1DNA and puromycin DNA as probes;

FIG. 98 shows the results of two-color FISH analysis of CHO (HPRT-; MAC4, hCHr7M-loxP-tel) clones using mouse Cot-1DNA and human Cot-1DNA as probes;

FIG. 99 shows the construction of mouse artificial chromosome MDR1-MAC obtained by transposing and cloning a MAC4 vector at 210kb around the human MDR1 gene region (AC 005045-human MDR1 gene-AC 003083);

FIG. 100 shows the results of two-color FISH analysis of CHO (MDR1-MAC, hCHr 7-. DELTA.MDR 1) clones using MDR1-BAC (RP11-784L5) (CHORI) DNA and mouse Cot-1DNA as probes.

Detailed Description

The present invention will be described in more detail below.

As described above, the first aspect of the present invention is characterized by comprising a natural centromere derived from a mouse chromosome, a long-arm fragment derived from a mouse chromosome, which is obtained by deleting the distal end of the long arm from the long-arm part of a mouse chromosome in the vicinity of the centromere, and a telomere sequence, and being stably carried in cells and tissues of a mammal.

The term "natural centromere derived from mouse chromosome" as used herein refers to the whole centromere (intact centromere) of any mouse chromosome. Thus, such centromeres do not include centromeres of chromosomes of other animal species and constructs having centromerfunction derived accidentally or artificially using a portion of the mouse chromosomal centromere sequence.

The "mouse artificial chromosome" or "mouse artificial chromosome vector" used in the present specification is an artificial chromosome constructed by a top-down method, not an artificial chromosome constructed by a bottom-up method. The top-down method refers to a method of constructing an artificial chromosome vector having a natural centromere by deleting a gene region from a natural chromosome through chromosome modification. The bottom-up method is a method for constructing an artificial chromosome having a centromere function by obtaining a part of a centromere sequence as a cloned DNA and transfecting it into a mammalian cell.

The "mouse chromosome-derived long-arm fragment obtained by deleting the distal end of the long arm from the long-arm part of the mouse chromosome near the centromere" as used herein means a long-arm fragment obtained by deleting the long-arm part near the centromere so as to remove the endogenous gene in the long arm of the mouse chromosome, since it is preferable that the vector of the present invention is stably carried in the cells or individual tissues of the mouse and the influence of the endogenous gene is excluded as much as possible so as not to inhibit the individual production and progeny transmission of the mouse. This means a long-arm fragment obtained by deletion at a long-arm site close to the centromere so that at least 99.5%, preferably at least 99.7%, more preferably at least 99.8%, most preferably 99.9 to 100% of the total endogenous genes (number) are removed.

The term "DNA" as used herein is intended to apply to all kinds of DNA nucleic acids including genes or gene loci, cDNAs, and chemically modified DNAs unless otherwise specified.

The term "carrying rate" as used herein refers to the proportion of cells containing artificial chromosomes present in cultured cells or in tissue cells of mice.

The "stably carrying" of the chromosomal vector of the present invention means that the chromosomal vector is not easily caused to fall off at the time of cell division, that is, can be stably carried in a cell even after division, and therefore, the chromosomal vector can be efficiently transferred to daughter cells or daughter mice by progeny.

The mouse chromosome may be any of mouse chromosomes 1to 19, X and Y, but is preferably any of chromosomes 1to 19. In the examples described below, chromosome 11 is exemplified, but as long as the above-described structure is provided, mouse artificial chromosome vectors can be similarly prepared even for other chromosomes.

In the case of an artificial chromosome vector derived from a mouse chromosome 11 fragment, the above-mentioned long-arm fragment is not limited, and is composed of, for example, a long-arm fragment in which a region more distal than the long arm of chromosome 11 is deleted, such as AL671968 or BX572640 (located closer to the centromere side than AL671968), CR954170 (located closer to the centromere side than AL671968 and BX 572640), or AL713875 (located closer to the centromere side than AL 671968). Alternatively, the long-arm fragment is DT40(MAC) in the present specification, and may contain a mouse artificial chromosome contained in deposited cell line DT40B6bT-1(FERM BP-11128) as a basic structure (see FIGS. 1, 3 and 4). In the case of an artificial chromosome vector derived from, for example, mouse chromosome 15 fragment, the long-arm fragment is not limited, and is composed of, for example, a long-arm fragment in which a region more distal than the position of AC121307, AC161799, or the like is deleted. In the case of an artificial chromosome vector derived from a mouse chromosome 16 fragment, the above-mentioned long-arm fragment is not limited, and is composed of, for example, a long-arm fragment in which a region more distal than the position of AC127687, AC140982, or the like is deleted. These basic structures also contain a DNA sequence insertion site such as loxP for inserting a foreign DNA or gene (see, for example, MAC1, MAC2, MAC3, MAC4, etc.; FIGS. 1, 3, and 4).

The vector of the present invention may contain a site for insertion of a foreign DNA or gene sequence, and thus, by inserting a target foreign DNA or gene into the site, the target foreign DNA or gene can be expressed when the vector is introduced into any cell, and thus, the vector can be applied to protein production, screening of therapeutic drugs, drug metabolism tests, functional analysis of DNA, induction of iPS cells, gene therapy, production of useful non-human animals, and the like (for example, refer to CYP3A-MAC, GFP-MAC, and the like; FIGS. 2 and 5).

The vectors of the invention also modify mouse chromosomes and use the natural centromeres derived from mice directly in making the vectors. As a conventionally known mouse artificial chromosome vector, there are known mammalian artificial chromosomes (referred to as Aces and SATAC) based on satellite DNA prepared using a part of the centromere sequence, but a mouse artificial chromosome prepared using the entire centromere of a mouse chromosome has not been prepared so far. In addition, the mammalian artificial chromosomes are not uniformly carried in tissues of individual mice and are unstable as in the HAC vector (Co Do et al, Chromosome Res.2000; 8 (3): 83-91).

Useful and unexpected characteristics of the carrier of the present invention include the following: since the gene is stably carried in a cell by increasing the carrying rate in a cell or an individual tissue of a mammal including a rodent such as a mouse, a rat, a hamster or the like, a target gene (group) can be stably carried for a long period of time, the gene can be uniformly expressed between rodent individuals or tissues for a long period of time, and the efficiency of individual generation and progeny transmission of rodents differentiated through pluripotent cells (e.g., ES cells, iPS cells or the like) can be improved. Interesting characteristics compared with Human Artificial Chromosomes (HACs) include a very low HAC carrier rate of less than 20% in blood system tissues, very little tissue heterogeneity, and a carrier rate of 90% or more in any post-test tissue (e.g., tissues derived from liver, intestine, kidney, spleen, lung, heart, skeletal muscle, brain, or bone marrow).

Defining:

definitions of terms used in the present specification include the following meanings in addition to the general meanings used in the industry.

In the present specification, the term "mouse artificial chromosome" or "mouse artificial chromosome vector" refers to an artificial chromosome having the above-described characteristics, which is prepared from a mouse-derived chromosome fragment as exemplified above, and which is constructed by the top-down method, not the bottom-up method. Repeating the above, the top-down method refers to a method of constructing an artificial chromosome vector having a natural centromere by deleting a gene region from a natural chromosome through chromosome modification. On the other hand, the bottom-up method is a method of constructing an artificial chromosome having a centromere function by obtaining a part of a centromere sequence as a cloned DNA and transfecting it into a mammalian cell. The artificial chromosome can be stably replicated and distributed as a chromosome that is independent of the original chromosome in the cell to be introduced. The mouse-derived chromosome fragment is a fragment of any of mouse chromosomes 1to 19, X and Y (long-arm fragment in which at least 99.5% of the total endogenous gene elements of the long arm are deleted), and includes a long-arm fragment in which the distal end of the long arm is deleted from a position of the long arm of the mouse chromosome near the centromere as defined above. The artificial chromosome of the present invention can be prepared by a method of preparing an artificial chromosome from a mouse chromosome 11 fragment as described in the examples described later, particularly in examples 1to 5, with reference to FIGS. 1to 4. The preparation of mouse artificial chromosomes from other chromosome fragments can be performed in exactly the same manner.

Sequence information of mouse chromosomes can be obtained from chromosome databases of DDBJ/EMBL/GenBank, Santa Cruz Biotechnology, Inc. and the like.

The "long arm" of the chromosome in the present specification means a chromosomal region containing a gene region from the centromere side of the mouse chromosome. On the other hand, there are few short arms in mouse chromosomes.

The "distal end" in the present specification means a region distant from the centromere (i.e., telomere side). In contrast, the region near the centromere (i.e., the centromere side) is referred to as the "proximal end". The distal end of the long arm means a region located closer to the telomere than the specific part of the long arm, and the proximal end of the long arm means a region located closer to the centromere than the specific part of the long arm. The specific site is a site where at least 99.5%, preferably at least 99.7%, more preferably at least 99.8%, most preferably 99.9 to 100% of the total endogenous genes (number) present in the long arm of one chromosome derived from a mouse are deleted.

The term "carrying rate" as used herein refers to the proportion of cells in which artificial chromosomes are present among cultured cells or tissue cells of a mouse.

The "insertion site of a DNA sequence" in the present specification refers to a site in an artificial chromosome into which a DNA (including a gene) sequence of interest can be inserted, for example, a recognition site of a site-specific recombinase or the like. Such recognition sites are not limited and include, for example, loxP (Cre recombinase recognition site), FRT (Flp recombinase recognition site),And(recombinase recognition sites), R4attB and R4attP (R4 recombinase recognition sites), TP901-1attB and TP901-1attP (TP901-1 recombinase recognition sites), or Bxb1attB and Bxb1attP (Bxb1 recombinase recognition sites), and the like.

The "site-specific recombinase" in the present specification is an enzyme that specifically causes recombination of a target DNA sequence at a recognition site of the enzyme. Examples thereof are Cre integrase (also referred to as Cre recombinase),Integrase, R4 integrase, TP901-1 integrase, Bxb1 integrase and the like.

The "telomere sequence" in the present specification is a natural telomere sequence of the same or different species, or an artificial telomere sequence. Herein, the homogeneous species refers to an animal that is the same species as a mouse from which a chromosome fragment of the artificial chromosome vector is derived, and the heterogeneous species refers to a mammal (including a human) other than the mouse. The term "artificial telomere sequence" refers to a sequence having a telomere function, which is artificially produced, such as a (TTAGGG) n sequence (n is a repeat). The introduction of a telomere sequence into an artificial chromosome can be carried out by telomere truncation (replacement of a telomere sequence) as described in, for example, International publication WO 00/10383. Telomere truncation may be used to shorten chromosomes in the production of artificial chromosomes of the invention.

The term "foreign gene" or "foreign DNA" as used herein refers to a gene or DNA to be inserted into a vector at a gene insertion site of the vector, which is a target gene or DNA carried in the vector, and is not originally present in a target cell and is to be expressed in the target cell, or a sequence thereof.

The term "mammal" as used herein includes primates such as humans, monkeys and chimpanzees, rodents such as mice, rats, hamsters and guinea pigs, ungulates such as cows, pigs, sheep and goats, but is not limited to these animals.

"embryonic stem cells" or "ES cells" in the present specification are stem cells established from the inner cell mass of a blastocyst derived from a fertilized egg of a mammal, which are equipped with differentiation pluripotency and semi-permanent proliferation ability (M.J.Evans and M.H.Kaufman (1981) Nature 292: 154-. Cells that have properties equivalent to those of the cells and are artificially induced by reprogramming of somatic cells are "induced pluripotent stem cells" or "iPS cells" (K.Takahashi and S.Yamanaka (2006) Cell 126: 663-676; K.Takahashi et al (2007) Cell 131: 861-872; J.Yu et al (2007) Science 318: 1917-1920).

Preparation and application of mouse artificial chromosome vector:

the preparation of the mouse artificial chromosome vector of the present invention and its use will be described below. Specifically, the procedure is described in examples 1to 5 (FIGS. 1to 4) described later.

(1) Preparation of mouse artificial chromosome vector

The artificial chromosome vector of the present invention can be prepared by a method comprising the following steps (a) to (c):

(a) obtaining cells carrying mouse chromosomes;

(b) a step of deleting the distal long-arm end of mouse chromosome so as not to contain most (99.5% to 100%) of endogenous genes (number) and

(c) inserting 1 or more DNA sequence insertion sites into the proximal end of the long arm. Here, the order of the steps (b) and (c) may be reversed.

A step (a):

to prepare the artificial chromosome vector of the present invention, first, cells carrying mouse chromosomes are prepared. For example, a mouse embryonic fibroblast (mCHr11-BSr) which is a mouse fibroblast cell introduced with a mouse chromosome labeled with a drug resistance gene (e.g., blasticidin S region gene (BSr)) and a mouse A9 cell (ATCC VA20110-2209) which is a G418 resistance gene neo gene (i.e., mouse A9(neo) are cell-fused, and the fusion can be prepared from a mouse A9 hybrid cell which is a mouse A9x mouse embryonic fibroblast (neo; mCHr11-BSr) which carries a mouse chromosome labeled with a drug resistance gene and the chromosome is transferred into a cell having a high homologous recombination rate. Mouse fibroblasts can be obtained based on literature-described methods, for example, can be established by mice of the C57B6 system available from clean, japan. As the cells having a high homologous recombination rate, for example, chicken DT40 cells (Dieken et al, Nature Genetics, 12: 174-182, 1996) can be used. The transfer can be carried out by a known chromosome transfer method, for example, micronuclear cell fusion method (Koi et al, Jpn. J. cancer Res., 80: 413-418, 1973).

A step (b):

in cells carrying a single chromosome derived from a mouse, the long arm distal end of the mouse chromosome is deleted. At this time, it is important to construct an artificial chromosome that lacks (or removes or deletes) most of the endogenous gene present on the long arm and carries the mouse centromere. This determines the position of the cleavage in such a way that at least 99.5%, preferably at least 99.7%, more preferably at least 99.8%, most preferably 99.9-100% of the total endogenous genes present on the long arm are deleted (or removed or deleted). Thus, the introduced artificial chromosome is stably carried in a cell, tissue or individual derived from a mammal such as a rodent, preferably a mouse, with a high carrying rate, and can be used for accurate analysis of a target gene (group), production of a substance, or the like. The deletion of the endogenous gene can be carried out, for example, by telomere truncation as described in WO 00/10383. Specifically, a targeting vector carrying an artificial telomere sequence is constructed in a cell carrying a mouse chromosome, and a clone in which an (artificial) telomere sequence is inserted at a desired position on the chromosome by homologous recombination is obtained, whereby a deletion variant can be obtained by telomere truncation. That is, the desired position (or site) is a cleavage site at the distal end of the long arm to be deleted, and the artificial telomere sequence is substituted or inserted at this position by homologous recombination to delete the distal end of the long arm. This position can be set as appropriate by the design of the target sequence when constructing the targeting vector. For example, in the examples described below, a target sequence was designed based on the DNA sequence of AL671968(GenBank accession number) of the long arm of mouse chromosome 11, and the target sequence was set so as to cause telomere truncation at the telomere side of the target sequence (see fig. 9). Thus, a mouse chromosome 11 fragment in which most of the endogenous gene is deleted can be obtained. Telomere truncation can be similarly performed in the case of other chromosomes.

A step (c):

as a site for inserting a DNA sequence, a recognition site for a site-specific recombinase can be preferably inserted. That is, it is known that a certain enzyme recognizes a specific recognition site and specifically causes recombination of DNA at the recognition site, and the mouse artificial chromosome vector of the present invention utilizes a vector comprising such an enzyme and the recognition site of the enzymeA system for inserting and carrying a target gene or DNA sequence. Examples of such a system include: system of Cre enzyme derived from bacteriophage P1 and its recognition site, i.e., loxP sequence (Cre/loxP system; B.Sauerin Methods of Enzymology; 1993, 225: 890-900), System of Flp enzyme derived from budding yeast and its recognition site, i.e., FRT (Flp Recombination target) sequence (Flp/FRT system), System of Streptomyces phage derivedIntegrase and its recognition sitea system of attB/attP sequence 6, a system of R4 integrase and its recognition site, that is, R4attB/attP sequence, a system of TP901-1 integrase and its recognition site, that is, TP901-1attB/attP sequence, a system of Bxb1 integrase and its recognition site, that is, Bxb1attB/attP sequence, and the like, and the system is not limited to the above systems as long as it can function as a DNA sequence insertion site.

For insertion of the recognition site of such a site-specific recombinase, a known method such as a homologous recombination method can be used, and the position and number of insertion into the proximal end of the long arm and the proximal end of the short arm can be appropriately set.

In the present invention, a single kind of recognition site or different kinds of recognition sites may be inserted. By setting the recognition site, the insertion position of the foreign gene or the foreign DNA can be specified, and therefore, the insertion position is constant and no unexpected position effect (position effect) is received. In the case of the mouse artificial chromosome as exemplified in examples described later, the gene inserted into the loxP sequence, which is the recognition site of the site-specific recombinase inserted into BX572640 on mouse chromosome 11, can be specifically expressed (fig. 11, 20, and 24).

In the mouse artificial chromosome vector having an insertion site for a DNA sequence of the present invention, it is preferable to reserve the insertion site for a gene of interest or a DNA sequence and previously insert a reporter gene. The reporter gene is not particularly limited, and examples thereof include: fluorescent proteins (e.g., green fluorescent protein (GFP or EGFP) gene, Yellow Fluorescent Protein (YFP), etc.), marker protein-encoding DNA, β -galactosidase gene, luciferase gene, etc., but GFP or EGFP is preferred.

The mouse artificial chromosome vector of the present invention may further contain a selection marker gene. The selection marker is effective in screening cells transformed by the vector. Examples of the selectable marker gene include either or both of a positive selectable marker gene and a negative selectable marker gene. The positive selection marker gene includes drug resistance genes such as neomycin resistance gene, ampicillin resistance gene, blasticidin s (bs) resistance gene, puromycin resistance gene, gentamicin (G418) resistance gene, hygromycin resistance gene, and the like. The negative selection marker gene includes, for mutexample, a herpes simpl mutex virus thymidine kinase (HSV-TK) gene, a diphtheria toxin A fragment (DT-A) gene, and the like. Typically HSV-TK may be used in combination with ganciclovir or acyclovir (シクロビル).

As a method for inserting a reporter gene, a target foreign gene or DNA into the mouse artificial chromosome vector of the present invention, homologous recombination can be preferably used. The homologous recombination can be carried out using a targeting vector obtained by ligating a DNA cassette to be inserted between the nucleotide sequences of the 5 '-side region and the 3' -side region (about 1to 4kb, preferably about 2 to 4kb, respectively) at the insertion position on the mouse chromosome and the same two sequences (5 '-arm (arm) and 3' -arm), and examples of vectors used for this purpose include plasmids, phages, cosmids, viruses, and the like, preferably plasmids.

The mouse artificial chromosome produced by the above method contains a mouse-derived chromosome fragment containing a natural centromere, a long-arm fragment lacking at least 99%, preferably at least 99.5%, of an endogenous gene, and, if present, a short arm, and an artificial telomere sequence. The centromere is the entire centromere structure of mouse chromosome used for the production of artificial chromosome.

An example of the mouse artificial chromosome vector of the present invention is a mouse artificial chromosome vector prepared in the examples described later, in which the distal end of the long arm is deleted in AL671968 in mouse chromosome 11 (fig. 1, 3, 4, 9, and 10). This vector contains, as a basic structure, the mouse artificial chromosome contained in DT40(MAC) in this specification, i.e., deposited cell line DT40B6bT-1(FERM BP-11128). Since it has a basic structure, a DNA sequence insertion site, a selection marker gene, a foreign gene (or DNA), and the like as described below can be inserted into the DNA structure.

The mouse artificial chromosome vector preferably contains 1 or more DNA sequence insertion sites, for example, recognition sites for a site-specific recombinase (e.g., loxP sites which are Cre enzyme recognition sites) (FIGS. 1, 3, 4, 11, 20, and 24). Here, the recognition site of the site-specific recombinase is, for example, a loxP site of GFP-PGKneo-loxP-3 'HPRT type, a loxP site of 5' HPRT-loxP-hyg type, PGKneo-loxP-3 'HPRT type, or a loxP site of GFP-5' HPRT-loxP-PGKhyg type, but is not limited thereto. Here, GFP is a green fluorescent protein gene, PGKneo is a phosphoglycerate kinase promoter/neomycin resistance gene cassette, HPRT is a hypoxanthine guanine phosphoribosyl transferase gene, and hyg is a hygromycin resistance gene.

The mouse artificial chromosome vector may further contain a reporter gene and a selection marker gene (e.g., a positive selection marker gene and a negative selection marker gene). The vector may also contain a target foreign gene or DNA sequence.

The mouse artificial chromosome vector of the invention has the same advantages as the existing artificial chromosome vector: 1) is not inserted into the host chromosome but is maintained independently, and therefore, does not disrupt the host gene; 2) stably carried at a certain copy number (which may be multiple (multiple) copies) and under the physiological expression control of the host cell, and thus, does not cause over-expression or disappearance of expression of the inserted gene; 3) since the size of the DNA that can be introduced is not limited, the portability of rodent cells or rodent individuals is improved compared to conventional artificial chromosomes, and stable expression of the introduced gene over a long period of time is achieved, and the offspring transmission rate is improved, in addition to the introduction of the gene or genes/subtypes that contain the expression regulatory region, and thus, the efficiency of production of transgenic mice is improved, (4) there is little variation between tissues after the introduction of the vector, that is, the portability is 90% or more for blood system tissues with a portability of less than 20% even in all tissues, and even in the case of HAC.

(2) Introduction of foreign genes or DNA

A foreign gene or DNA may also be introduced into the mouse artificial chromosome vector of the present invention.

The size of the foreign gene or DNA sequence is not particularly limited, and may be 20kb or less, or may exceed 20kb, for example, 50kb or more, 100kb or more, 200kb or more, 500kb or more, 700kb or more, 1Mb or more, 10Mb or more, 20Mb or more, 30Mb or more, 40Mb or more, or 50Mb or more. The vector of the present invention can carry a foreign DNA (chromosome fragment) of 1Mb or more, which is a size difficult for artificial chromosome vectors such as BAC, PAC, YAC and the like, as in the case of HAC vectors. Therefore, the vector of the present invention can carry a large foreign gene or DNA fragment of 200kb or more, for example, 1Mb or more, stably and at a higher carrying rate (90% or more) than HAC in mammalian cells, tissues or non-human animal individuals, preferably rodent cells, tissues or individuals, as described above.

As one embodiment of the present invention, the present invention provides a vector capable of stably maintaining a foreign gene or DNA having a large size of 200kb or more in a rodent cell or a rodent individual at a carrying rate of 90% or more, and a method for producing the vector.

The foreign gene or DNA is a nucleic acid sequence to be introduced from the outside into a target cell, is not particularly limited, and is a gene or DNA derived from any biological species or from any tissue or cell, preferably a mammalian gene or DNA, and more preferably a human gene or DNA. Such genes or DNAs include genes or DNAs encoding polypeptides such as cytokines, hormones, growth factors, nutritional factors, hematopoietic factors, immunoglobulins, G protein-coupled receptors, enzymes and the like, therapeutic genes or DNAs related to various diseases including tumors, muscular dystrophy, hemophilia, neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's chorea, Parkinson's disease and the like), autoimmune diseases, allergic diseases, genetic diseases and the like, genes (groups) of (human) drug metabolizing enzymes or DNA (human) drug metabolism-related genes, DNAs of long or short arms of human chromosomes, and (human) genomic libraries, but are not limited thereto.

Cytokines include, for example, interferons (e.g., IF-alpha, IF-beta, IF-gamma, etc.), interleukins (e.g., IL-1, IL-2, IL-4, IL-6, IL-11, IL-12, etc.), tumor necrosis factors (e.g., TNF-alpha, TNF-beta), TGF-beta family proteins (e.g., Bone Morphogenic Proteins (BMPs), etc.), and the like.

Hormones include, for example, growth hormone, human chorionic gonadotropin (hCG), human placental lactogen (hPL), human pituitary gonadotropin, Thyroid Stimulating Hormone (TSH), luteinizing hormone releasing factor, insulin, glucagon, somatostatin, prolactin, and the like.

Growth factor or trophic factor classes include, for example, insulin-like growth factor, brain-derived neurotrophic factor (BDNF), albumin-fused ciliary neurotrophic factor, platelet-derived neurotrophic factor (PDNF), transforming growth factor, Nerve Growth Factor (NGF), TNF growth factor, and the like.

The coagulation and hemolysis factors include, for example, factor VII, factor VIII, factor X, t-PA and the like.

Hematopoietic factors include, for example, erythropoietin, (granulocyte) colony stimulating factor, thrombopoietin, and the like.

The immunoglobulin includes, for example, human antibodies to various antigens, recombinant antibodies such as humanized antibodies, chimeric antibodies and synthetic antibodies, and the like.

G protein-coupled receptors include adrenergic receptors, muscarinic acetylcholine receptors, adenosine receptors, GABA receptors (type B), angiotensin receptors, cholecystokinin receptors, dopamine receptors, glucagon receptors, histamine receptors, olfactory receptors, opioid receptors, secretin receptors, somatostatin receptors, gastrin receptors, P2Y receptors, and the like.

Enzymes include, for example, asparaginase, superoxide dismutase, uricase, streptokinase, dopamine synthase, adenosine deaminase, and the like.

Therapeutic genes associated with various diseases including tumors, muscular dystrophy, neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's chorea, Parkinson's disease, etc.), autoimmune diseases, allergic diseases, genetic diseases, etc., include genes such as dystrophin gene, IL-12 gene, TNF- α gene, tumor suppressor gene, dopamine synthase gene, genetic enzyme deletion ( Yushima enzyme), etc.

Drug-metabolizing enzymes are enzymes involved in metabolic reactions for decomposing and discharging foreign substances such as drugs and poisons, and include enzymes involved in first-phase reactions (oxidation, reduction, hydrolysis) and enzymes involved in second-phase reactions (binding). The enzymes involved in the first phase reaction include, for example, known enzymes such as cytochrome P450 ("CYP"), specifically, CYP1A, CYP1B, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2J, CYP3A, CYP4A, CYP4B, and subfamilies thereof, and CES. As the CYP subfamilies, for example, the subfamilies of CYP3A include CYP3a4, CYP3a43, CYP3a5, CYP3a7 and the like, and the subfamilies of CYP2C include CYP2C8, CYP2C9, CYP2C18, CYP2C19 and the like. That is, CYP3A-MAC described in examples described later refers to the CYP3A cluster and is composed of CYP3a4, CYP3a43, CYP3a5, and CYP3a 7. On the other hand, enzymes involved in the second phase reaction (binding) include, for example, UGT1 and UGT 2.

The drug metabolism-related gene includes, for example, a gene encoding a transporter, a gene encoding a nuclear receptor, and the like. Examples of genes encoding transporters are MDR1, MDR2, MRP2, OAT, OATP, OCT, BCRP, etc., and examples of genes encoding nuclear receptors are PXR, AhR, CAR, PPAR α, etc.

As described above, the foreign DNA sequence related to drug metabolism that can be introduced into the vector of the present invention may contain a sequence of at least 1 gene or a sequence of at least 2 genes selected from the group consisting of a gene encoding an enzyme related to the first phase reaction, a gene encoding an enzyme related to the second phase, a gene encoding a transporter protein, and a gene encoding a nuclear receptor.

At least 1 insulator sequence may be present near or on both sides of the insertion site of the foreign gene or foreign DNA in the mouse artificial chromosome vector of the present invention. Insulator sequences have enhancer blocking effects (i.e., adjacent genes are not affected by each other) or chromosome boundary effects (distinguishing regions where gene expression is guaranteed from regions where gene expression is inhibited). Such sequences include, for example, human beta globin HS 1-HS 5, chicken beta globin HS4, and the like.

The introduction of the foreign gene or DNA can be carried out by using the system of site-specific recombinase as exemplified above inserted as the insertion site of the DNA sequence described above. For example, a targeting vector carrying a loxP site which is a recognition site of Cre enzyme and a foreign gene or DNA or a chromosome fragment carrying a foreign gene or DNA into which a loxP site which is a recognition site of Cre enzyme is inserted is constructed, Cre enzyme is expressed in cells carrying the mouse artificial chromosome vector of the present invention, and thus a foreign gene or DNA can be introduced by site-specific recombination with the targeting vector or the chromosome fragment in the loxP site.

A site-specific recombinase may also be inserted into the mouse artificial chromosome vector of the present inventionThe recognition site (for example, circular DNA such as loxP site, FRT site, etc., can also be used to insert cloned DNA using existing vectors such as plasmid using Escherichia coli as host or circular YAC using yeast as host. the preferred loxP site is a wild-type sequence derived from P1 phage, and the insertion reaction of the circular insert (インサ - ト) into the loxP site on the artificial chromosome vector obtained by Cre enzyme is reversibleThe recognition site of integrase, i.e., a combination of attB/attP sequences, etc., can also be used to construct a system in which a plurality of circular inserts are inserted in order without causing reverse reaction.

(3) Transfer of mouse artificial chromosome vector into cell and production of non-human animal

The mouse artificial chromosome vector of the present invention or the mouse artificial chromosome vector of the present invention containing a foreign gene or DNA can be transferred or introduced into any cell. The method for engraftment or introduction includes, for example, a micronuclear cell fusion method, a lipofection method, a calcium phosphate method, a microinjection method, an electroporation method, etc., and a preferable method is a micronuclear cell fusion method.

The micronucleus cell fusion method is a method of transferring a mouse artificial chromosome vector of the present invention into other desired cells by micronucleus fusion of the cell having micronucleus-forming ability (e.g., mouse a9 cell) and the other cells. Cells having micronucleus-forming ability are treated with a polyploid inducer (e.g., colchicine \ colchicine, etc.) to micronucleus the cells, and after treatment to form a micronucleus by cytochalasin treatment, cell fusion with desired cells is performed.

The cells into which the vector can be introduced include animal cells, preferably mammalian cells containing human cells, such as germ cells including oocytes and spermatids, stem cells including Embryonic Stem (ES) cells, sperm stem (GS) cells and somatic stem cells, somatic cells, fetal cells, adult cells, normal cells, diseased cells, primary cultured cells, secondary cells, cell lines, and the like. The stem cells include, for example, ES cells, Embryonic Germ (EG) cells, Embryonic Carcinoma (EC) cells, mGS cells, pluripotent stem cells such as human mesenchymal stem cells, Induced Pluripotent Stem (iPS) cells, nuclear transfer-derived embryonic stem (ntES) cells, and the like. Preferred cells are selected from the group consisting of somatic cells, non-human germ cells, stem cells and precursor cells derived from mammals, preferably rodents containing mice. When the cell is derived from a mammal such as a rodent, the vector of the present invention can be carried more stably in a cell or tissue of a mammal (for example, a rodent such as a mouse) into which the vector is introduced, i.e., the vector can be carried significantly less or can not be caused to fall off from the cell.

Examples of the cells include hepatocytes, intestinal cells, kidney cells, spleen cells, lung cells, heart cells, skeletal muscle cells, brain cells, bone marrow cells, lymphocytes, megakaryocytes, sperm, and ova.

The tissue is, for example, liver, intestine, kidney, spleen, lung, heart, skeletal muscle, brain, bone marrow, testis, ovary, etc.,

for ES cells, it is possible to take out an inner cell mass from a blastocyst of a fertilized egg of a subject animal and establish and maintain mitomycin C-treated mouse embryonic fibroblasts as a feeder layer (M.J. Evans and M.H. Kaufman (1981) Nature 292: 154-156).

iPS cells are cultured and subcultured by introducing a specific reprogramming factor (DNA or protein) into somatic cells (including somatic stem cells) in an appropriate medium, and thereby colonies are generated in about 3 to 5 weeks. Reprogramming factors are known to be, for example, a combination of Oct3/4, Sox2, Klf4, and c-Myc; a combination of Oct3/4, Sox2, and Klf 4; composed of Oct4, Sox2, Nanog and Lin28A combination of (a) and (b); or a combination of Oct3/4, Sox2, Klf4, c-Myc, Nanog and Lin28 (K.Takahashi and S.Yamanaka, Cell 126: 663-676 (2006); WO 2007/069666; M.Nakagawa et al, Nat.Biotechnol.26: 101-872 (2008); K.Takahashi et al, Cell 131: 861-872 (2007); J.Yu et al, Science 318: 1917-1920 (2007); J.Liao et al, Cell Res.18, 600-603 (2008)). An example of the culture includes introducing a strain of mouse embryonic fibroblasts (for example, STO) treated with mitomycin C as feeder cells into somatic cells (about 10) at a temperature of about 37 ℃ using a medium for ES cells on the feeder cell layer⁴～10⁵Cells/cm²) The culture is carried out. Feeder cells are not necessary (Takahashi, K., et al, Cell 131: 861-872 (2007)). The minimal medium may be, for example, Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 medium, or a mixed medium thereof, and as the ES cell medium, a mouse ES cell medium, a primate ES cell medium (Resprocell (リプロセル)), or the like can be used.

Since ES cells and iPS cells contribute to the reproductive line, a non-human animal (or a transgenic animal (excluding human) can be produced by a method comprising injecting these cells into blastocysts of embryos of mammals of the same species from which the cells are derived, into which the mouse artificial chromosome vector of the present invention containing a target gene or DNA has been introduced, and transplanting the embryos into the uterus of a foster mother to allow the embryos to grow. Further, by mating the male and female transgenic animals thus obtained, homozygous animals and further offspring animals thereof can be produced.

By introducing a foreign gene or DNA such as a human antibody gene, a gene for treating a disease, a gene related to drug metabolism into a differentiated pluripotent cell such as an ES cell or an iPS cell or another cell group via the mouse artificial chromosome vector of the present invention, a cell or a non-human animal capable of producing a human antibody, a cell capable of producing a protein for treating a disease, or a disease model non-human animal such as a disease related to drug metabolism can be produced.

In such a non-human animal, it is also sometimes preferable that an endogenous gene corresponding to an exogenous gene contained in the mouse artificial chromosome vector is disrupted or that the expression of the endogenous gene is reduced. The disruption method may be a gene targeting method. The method of reducing the expression of an endogenous gene may be an RNAi method. Examples of such foreign genes include: drug metabolism-related genes, human antibody genes, and the like. The non-human animal in which the endogenous gene is disrupted can be produced by further mating animals heterozygously deleted for the endogenous gene obtained by mating a chimeric non-human animal or its offspring carrying the mouse artificial chromosome vector containing the exogenous gene with a corresponding chimeric animal or offspring deleted for each cluster of the endogenous gene.

Cells carrying the mouse artificial chromosome vector and transgenic non-human animals can be prepared by the method. Specific examples of the non-human animal include rodents such as mice or rats carrying a mouse artificial chromosome vector.

That is, the present invention provides a cell and a non-human animal, which are characterized by carrying a mouse artificial chromosome vector.

Furthermore, the cells, tissues or organs obtained from the non-human animals of the present invention can also be used for the production of cell lines that produce proteins by expression of foreign genes from these cells.

(4) Method for producing useful protein

The present invention also provides a method for producing a protein, which comprises: cells carrying a mouse artificial chromosome vector containing an expressible foreign DNA sequence are cultured, and the protein encoded by the DNA produced is recovered.

Examples of the protein include proteins and polypeptides useful in industrial fields such as the medical field and the agricultural field. Appropriate cells are transformed or transfected by inserting a DNA encoding these proteins or polypeptides into a mouse artificial chromosome vector so that the DNA can be expressed in the presence of a promoter (and optionally an enhancer). The resulting cells are cultured to express the DNA to produce the protein or polypeptide, which is recovered from the cells or the culture medium.

As the cells, in addition to mammalian cells, insect cells such as Sf cells, avian cells, yeast cells, plant cells and the like can be used.

The culture conditions including the medium may be selected depending on the type of the cells, and known conditions may be used as the culture conditions. For example, the culture medium for animal cells includes MEM medium, DMEM medium, Ham's F12 medium, Eagle's MEM medium, Iscove's EME medium, RPMI1640 medium, a mixed medium thereof, and the like.

The recovery (or separation) of the protein or polypeptide can be carried out by a known method such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, HPLC, FPLC, salting-out method, ammonium sulfate precipitation, organic solvent precipitation, ultrafiltration, crystallization, or the like, alone or in combination.

(5) Method for producing human antibody

The present invention also provides a method for producing a human antibody, which comprises: the above-mentioned non-human animal carrying the mouse artificial chromosome vector containing the human antibody gene is used to produce a human antibody and the human antibody is recovered.

The human antibody gene is a gene encoding any one of human IgG, IgM, IgA, IgD, and IgE, or any one of subclasses of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. Preferred human antibody genes are the IgG class and subclasses thereof.

Human antibodies consist of 2 heavy chains (H) of identical sequence and 2 light chains (L) of identical sequence, both H and L chains consisting of variable and constant regions. The variable regions of the human H chain and L chain were each composed of 3 hypervariable regions (in the order of CDR1, CDR2, and CDR3 from the N-terminal side to the C-terminal side) and 4 framework regions (in the order of FR1, FR2, FR3, and FR4 from the N-terminal side to the C-terminal side), and the specificity of the antibody was determined from the respective 3 CDR sequences of the human H chain and L chain.

In the case of human IgG antibodies, consisting of a μ chain as a heavy chain and a λ chain or a κ chain as a light chain, these antibody chain genes are present on human chromosome 14, chromosome 22, and chromosome 2, respectively. As the human antibody gene used in the present invention, a human chromosome fragment containing each antibody locus is used, and these are inserted into different or the same mouse artificial chromosomes. The antibody gene sequence can be obtained from the database of NCBI (U.S.A.) and the like. This series of methods is an improvement of the technique described in, for example, japanese patent laid-open No. 2005-230020.

Non-human animals producing fully human antibodies chimeric non-human animals and their progeny animals having both H chain and L chain loci can be obtained by mating non-human animals carrying a mouse artificial chromosome vector containing a human mu chain locus with non-human animals of the same species carrying a mouse artificial chromosome vector containing a human lambda chain locus and/or a human kappa chain locus.

The human antibody can be produced in the non-human animal (e.g., rodent such as mouse) which is produced as described above and which can produce a fully human antibody, by a method comprising immunizing a specific antigen peptide or antigen polypeptide and isolating the human antibody from the blood of the animal.

Alternatively, a spleen of a non-human animal immunized with a specific antigen may be removed and fused with myeloma cells to obtain hybridoma cells producing a monoclonal antibody.

(6) Method for screening therapeutic substances

The present invention also provides a method for screening a substance effective for treating a disease, which comprises administering a candidate agent to the above-mentioned non-human animal as a model animal for the disease and evaluating the therapeutic effect of the agent.

The disease model non-human animal is an animal artificially created and having a disease caused by abnormality such as biological function abnormality due to defect or mutation of a certain protein, drug metabolism abnormality, or chromosome abnormality. Examples of model non-human animals with chromosomal abnormalities are, but are not limited to, animals with human chromosome 18 or 21 triploid disease.

Such non-human animals can be made by the following method: the method comprises preparing a gene or a chromosome fragment containing the above-mentioned abnormality in the gene or chromosome, inserting the gene or chromosome fragment into the mouse artificial chromosome vector of the present invention, introducing the gene or chromosome fragment into ES cells or iPS cells, injecting the cell into a blastocyst of a fertilized egg, and transplanting the cell into the uterus of a mother body of a non-human animal to produce the cell.

By administering the candidate drug to the non-human animal prepared as described above and evaluating the therapeutic effect of the drug, a substance effective for treating the disease can be screened.

The candidate drug is not limited, and examples thereof include low molecular weight compounds, high molecular weight compounds, (glyco) proteins, peptides, (phospho or glyco) lipids, and saccharides.

(7) Method for testing pharmacological action, metabolism or toxicity of drug or food

According to an embodiment of the present invention, there is provided a method for testing pharmacological effects and/or metabolism and/or toxicity of a drug or food, comprising administering the drug or food to the non-human animal or a cell, organ or tissue derived from the non-human animal carrying the mouse artificial chromosome vector containing a gene related to human drug metabolism, and measuring the pharmacological effects and/or metabolism and/or toxicity of the drug or food.

The present invention also provides a method for testing toxicity of a drug or food, which comprises culturing microsome or microsome fraction S9 obtained from the above-mentioned non-human animal carrying the mouse artificial chromosome vector containing a gene related to human drug metabolism together with cultured cells or bacteria and a drug or/and a food and measuring the (adverse) effect (e.g., mutation, etc.) of the drug or food on the cells or bacteria.

The human drug metabolism-related gene is the gene exemplified above. The method for producing a non-human animal is also the same as described above.

In the above-mentioned method using the above-mentioned non-human animal carrying the mouse artificial chromosome vector having the gene related to human drug metabolism, the pharmacological action, metabolism or toxicity of the drug or food can be determined, for example, by observing the state of the animal, or by testing the influence on organs and chromosomes.

In another method of the present invention, microsomes or microsomes fraction S9(9000g fraction, i.e., a fraction containing a plurality of enzymes that catalyze hydrolysis, reduction, oxidation, binding, and the like) obtained from the non-human animal is cultured together with cultured cells (particularly animal cells, preferably mammalian cells) or bacteria (preferably salmonella) in the presence of a drug and/or food. The toxicity of the drug or food to the cells can be detected by the Ames test or the micronucleus test. In the Ames test, toxicity is determined based on variation of Salmonella. In the micronucleus test, toxicity is judged based on chromosomal abnormalities in the nucleus. These assays are well known and can be used in the methods of the present invention.

The present invention will be described in further detail below with reference to examples, but the scope of the present invention is not limited to these specific examples.

Examples

[ example 1]

Construction of mouse artificial chromosome vector MAC

Mouse artificial chromosome MAC without endogenous genes was constructed by telomere truncation of mouse chromosomes [ DT40(B6bT) ] (fig. 1).

[A] Establishment of hybrid cell of A9 cell and mouse fibroblast (neo; mCHr11-BSr)

A mouse A9 hybrid cell carrying a mouse chromosome labeled with a drug resistance gene, namely, a mouse A9x mouse embryo fibroblast (neo; mCHr11-BSr), is established by fusing a mouse embryo fibroblast (mCHr11-BSr), which is a mouse fibroblast containing a mouse chromosome 11 labeled with a drug resistance gene (Bsr gene), with a mouse A9(neo) cell into which a G418 resistance gene, namely, a neo gene, has been inserted into a known mouse A9 cell. In order to introduce a mouse chromosome labeled with a drug resistance gene into chicken DT40 cells having a high homologous recombination frequency by the micronuclear cell fusion method, a mouse chromosome labeled with a drug resistance gene was introduced into mouse A9 cells known to have a high micronucleation rate by cell fusion.

[ A.1] cell fusion and isolation of double-agent resistant clones

Established from a mouse embryo of the C57B6 system available from CLEA, a mouse embryo fibroblast (mCHr11-BSr) which is a mouse fibroblast having a drug resistance gene (Bsr gene) inserted into its chromosome and a mouse A9(neo) which is a mouse A9 cell having a neo gene as a G418 resistance gene inserted into its chromosome were washed with PBS (-) to remove the cell surfaces, and then trypsin was added thereto to disperse the cells, which were suspended in a culture medium (10% FBS, DMEM) to obtain various cells 1 × 10⁶Simultaneously, a culture flask (25 cm) was placed²) The cultivation was carried out for one day. After washing the cell surface 2 times with PBS (-), 3ml of PEG (1: 1.4) solution [ 5g, PEG1000, cat: 165-09085, wako was dissolved in 6ml of serum-free DMEM, and 1ml of dimethyl sulfoxide was added thereto for filtration sterilization]The treatment was carried out for 1 minute and replaced with 3ml of PEG (1: 3) solution [ 5g, PEG1000, cat: 165-09085, wako was dissolved in serum-free DMEM 15ml and filter-sterilized]The treatment was carried out for 1 minute. After the PEG solution was aspirated, the cells were washed 3 times with serum-free DMEM, and cultured in a usual culture medium (10% FBS, DMEM) for one day. After washing the cell surface with PBS (-), cells were dispersed by adding trypsin, and cells suspended in a double selection medium (10% FBS, DMEM) containing G418 (800. mu.g/ml) and blasticidin S (4. mu.g/ml) were plated in plastic petri dishes for 2-3 weeks selection. A total of 3 resistant colonies obtained by 2 cell fusions were separated and proliferated, and analyzed as follows (clone name: mouse A9x mouse embryo fibroblast hybrid (neo; mCHr 11-BSr)).

[ A.2] selection of hybrid cells

[A.2.1]PCR

Genomic DNA was extracted from the double-drug resistant clones, and PCR was performed using the following primers to confirm mouse chromosomes carrying a marker for the drug resistance gene (Bsr gene).

Bsr R1: 5 'CATGTGGGAGCGGCAATTC 3' (SEQ ID NO: 1)

Bsr L1: 5 'TTGAGTGGAATGAGTTCTTCAATCG 3' (SEQ ID NO: 2)

For PCR, Gene Amp9600 manufactured by Perkin-Elmer was used as a thermal cycler, Ampli Taq Gold (Applied Biosystems) was used as Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after heat denaturation at 95 ℃ for 10 minutes, 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds and 72 ℃ for 30 seconds were carried out. For the results of PCR, 3 out of 3 clones were positive.

TABLE 1

[ A.2.2] Quinazine Hoechst double staining

For clones positive by the above PCR analysis, a quinacrine-Hoechst double staining was performed. For the quinacrine Hoechst double staining, first, a chromosome slide was immersed in 50ml of a meyer's solution (マキルベン) [ 11.18g of citric acid monohydrate and 13.29g of disodium hydrogen phosphate were dissolved in 1L of water and autoclaved ], and then, in 50ml of the meyer's solution, quinacrine [ cat: q2876, SIGMA ], the back surface of the chromosome slide was washed with tap water, and then immersed in a mekholdham solution, and Hoechst [ cat: b-2883] for 15 minutes, and then covered with a cover glass. As a result of observation with a fluorescence microscope, most of the 3 clones had a karyotype of 4n or more, where the normal karyotype was 2 n. In particular, it was found that in clone A9(21-B6B)7, mouse fibroblasts carrying a mouse chromosome labeled with a drug resistance gene (Bsr gene) and mouse A9 cells into which a neo gene as a G418 resistance gene was inserted were cell-fused one by one (FIG. 6).

TABLE 2

From the above results, the following conclusions can be drawn: in mouse A9x mouse embryonic fibroblast hybrid (neo; mCHr11-BSr), a labeled mouse chromosome was carried.

[B] Introduction of mouse chromosome labeled with drug resistance Gene into DT40 cells

The mouse chromosome marked with the drug resistance gene was introduced into chicken DT40 cells, namely DT40, from a mouse A9 hybrid cell, namely mouse A9x mouse embryonic fibroblast hybrid (neo; mCHr11-BSr), which contained the mouse chromosome marked with the drug resistance gene. In order to efficiently perform the identification of mouse chromosome number and the site-specific excision of chromosome caused by the insertion of artificial telomere, i.e., telomere truncation, and the insertion of DNA sequence insertion site, i.e., loxP sequence, into mouse chromosome by homologous recombination, the mouse chromosome marked with drug-resistant gene is introduced into chicken DT40 cell, i.e., DT40, which has high homologous recombination frequency, by micronuclear cell fusion method.

[ B.1] micronucleus cell fusion and isolation of drug-resistant clones

To efficiently perform chromosome identification and chromosome modification, mice were stainedIn vivo, chicken DT40 cells with high homologous recombination frequency, namely DT40, were transferred from A9 hybrid cell clone, namely A9x mouse embryo fibroblast hybrid (neo; mCHr11-BSr)7, and donor cells cultured with flask × 24, namely A9x mouse embryo fibroblast hybrid (neo; mCHr11-BSr)7, were confluent (confluent) 70% in each flask at 37 ℃ with 5% CO₂Under the conditions of (1) A fall ceramide treatment was carried out for 48 hours (fall ceramide 0.05. mu.g/ml, 20% FCS, DMEM). After the completion of the autumn acrylamide treatment, the medium (medium) in the flask was aspirated, and the flask was filled up to 9 days with cytochalasin B. The flask was inserted into a vessel dedicated to a large high-speed centrifuge (BECKMAN), warm water (34 ℃ C.) was added to such an extent that the flask was not covered, and centrifugation was carried out (Rortor ID10.500, 8,000rpm, 1 hour, 34 ℃ C.). After completion of centrifugation, cytochalasin B was recovered, and the particles in each flask were recovered in a 15ml tube using 2ml of serum-free medium DMEM. After filtration was performed gradually in the order of 8 μm → 5 μm → 3 μm filter, each tube was centrifuged (1,200rpm for 5 minutes R.T), and after the supernatant was aspirated, the particles in each tube were collected and collected, suspended in 5ml of serum-free medium DMEM, and centrifuged (2000rpm for 5 minutes).

DT40 was attached to 1 well of a 6-well plate (Nunc), 1 well was incubated overnight at 37 ℃ with 1.5ml of polylysine (SIGMA) adjusted to 50. mu.g/ml, thereby coating (コ - テイング), polylysine was recovered, the plate was washed with PBS (-), and about 1 × 10⁷DT40 cells were gently seeded in the plates with 2ml of serum free medium (DMEM). Plates were placed together in a centrifuge (Beckman) and centrifuged at 1200rpm for 3 minutes at 37 ℃ to make adherent DT 40.

The purified micronucleus cells were resuspended in 2ml of serum-free medium containing PHA-P (SIGMA), and gently seeded on adherent DT40 from which serum-free medium (DMEM) was removed. Plates were centrifuged at 1200rpm for 3 minutes at 37 ℃. The supernatant was removed and the PEG1000(Wako) [ 5g of PEG1000 was completely dissolved in serum-free DMEM medium, 1ml of dimethyl sulfoxide was added and filtration sterilization was performed ] solution was correctly fused with 1ml for 1 minute. Serum-free medium (DMEM) was washed 4 times with 4ml, pipetted with 3ml of ordinary DT40 medium to return the adherent DT40 to a floating state, and inoculated in 2-well 24-well plates at 37 ℃ for overnight culture. Blasticidin S was added to 1500. mu.g/ml, and selection and culture were carried out for 3 to 4 weeks. Total 2 resistant colonies obtained by primary micronucleus fusion were isolated and proliferated for subsequent analysis (clone name: DT40(mCHr11-BSr))

[ B.2] screening of drug-resistant clones

[ B.2.1] FISH analysis

The clone of DT40(mCHr11-BSr) obtained as described above was subjected to FISH analysis using mouse Cot-1DNA as a probe by the method described in Shinohara et al report (human molecular Genetics, 10: 1163-1175, 2001), and as a result, 95% of the mouse chromosomes per normal karyotype (2n) were 1 copy in DT40(mCHr11-BSr) -1, and the subsequent analysis was carried out (FIG. 7).

TABLE 3

[ B.2.2] identification of mouse chromosome marked with drug resistance Gene introduced into DT40

SKY-FISH was carried out by the method described in Kai et al (Cell Res, 19: 247-58, 2009) report, and it was found that the mouse chromosome introduced into the chicken DT40 cells was mouse chromosome 11 (FIG. 8).

[C] Site-specific cleavage of mouse chromosome 11 region AL671968 from chicken DT40 cells using distal telomere truncation

In the case where the amount of an endogenous gene other than a target gene introduced into a mouse artificial chromosome vector is small, it is necessary to reduce the influence on an experimental system, and it is necessary to leave as little as possible a gene that influences the mouse individual due to a change in the expression level of the gene, such as a imprinted gene in the endogenous gene, and therefore most of the long arm of the mouse is deleted.

[ C.1] telomere truncation vector preparation

The basic vector for the site-specific cleavage at the proximal short arm used was pBS-TEL/puro construct (コンストラクト) (Kuroiwa et al Nature Biotech 2002). A target sequence for homologous recombination was designed from the base sequence (AL671968) near the long arm of mouse chromosome 11 obtained from the GenBank database. Genomic DNA was extracted from DT40(mCHr11-BSr) as a template, and the sequences of primers used for PCR amplification of the homologous recombination target sequences are shown below.

m 1117L: 5'-CGAGGATCCCACATTGGTAGTCTTTTCACTGCCATCA-3' (SEQ ID NO. 3)

m 1117R: 5'-CGAGGATCCCCACTTAACTTTTCCAGGCTTACGGAGA-3' (Serial number 4)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The PCR product thus obtained was digested with BamHI (TAKARA) and separated and purified on agarose gel, and then cloned into the BamHI site of pB S-TEL/puro (vector name: pB S-TEL/puro _ MAC) after heat denaturation at 94 ℃ for 1 minute, followed by 35 cycles at 98 ℃ for 10 seconds and 65 ℃ for 8 minutes. The targeting vector, target sequence and chromosomal allele produced by homologous recombination are shown in FIG. 9.

[ C.2] screening of homologous recombinants

A vector having been cleaved site-specifically from the distal end of mouse chromosome 11 region AL671968 was transfected with pBS-TEL/puro _ MAC described above, and puromycin-resistant and blasticidin S-non-resistant clones were isolated and homologous recombinants were selected. As a result, it was confirmed that 5 clones (clone name: DT40(MAC)) capable of cleaving the mouse chromosome 11 region were obtained. The targeting vector, target sequence, and chromosomal allele produced by homologous recombination are shown in FIG. 9.

[ C.3] screening of drug-resistant clones by Monochromatic FISH analysis

FISH analysis using mouse Cot-1DNA as a probe was performed on 5 clones of DT40(MAC) obtained as described above by Shinohara et al (human molecular Genetics, 10: 1163-1175, 2001), and it was confirmed that the long-arm portion of mouse chromosome 11 was cleaved in the vicinity of the centromere in2 clones out of the 5 clones (FIG. 10).

TABLE 4

From the above results, the following conclusions can be drawn: mouse artificial chromosome MAC with the redundant long arms of mouse chromosomes excised can be constructed. Chicken DT40 cells carrying mouse artificial chromosome vector MAC, i.e., DT40(MAC) -1, were internationally deposited as identified name DT40B6bT-1 at the patent organism depositary center of the integrated human industrial technology institute of independent government institute of technology (center 6 of 1 st prefecture 1 st of 1 st bust of tokyo, ltd. ken., japan) on 5/14 th day in 21 years (2009) based on the provisions of the budapest treaty, and assigned deposit number FERM BP-11128.

EXAMPLE 2 construction of mouse Artificial chromosome vector MAC1

Mouse artificial chromosome vector MAC1 was constructed by inserting a GFP-PGKneo-loxP-3' HPRT-type loxP sequence as a DNA insertion sequence into mouse artificial chromosome MAC, and the stability of MAC1 in mouse ES cells was examined, and further, a progeny transfer mouse into which MAC1 was introduced was prepared, and the stability in individual tissues was examined.

[A] Insertion of GFP-PGKneo-loxP-3' HPRT type loxP sequence into mouse Artificial chromosome vector MAC

[ A.1] preparation of GFP-PGKneo-loxP-3' HPRT type loxP targeting vector

The basic plasmid for inserting the loxP site into DT40(MAC) was V913(Lexicon genetics). The DNA sequence of mouse chromosome 11 as the loxP insertion site was obtained from the GenBank database (BX 572640.9). Genomic DNA was extracted from the drug-resistant clones as a template, and the sequences of primers for amplification of two target sequences for homologous recombination are shown below.

m 115L: 5'-TGACAGAGAGCTTCCTCCTGCCTCTGTA-3' (Serial number 5)

m 115R: 5'-CTAAAGACCCTCATGCTCCTGTGTGGAA-3' (Serial number 6)

m 116L: 5'-GTTCAACCTGAGCTCCACATCATGCTC-3' (Serial number 7)

m 117R: 5'-CACTCTTTACCCCTCACCGCTAACCTTG-3' (Serial number 8)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 10 seconds and 68 ℃ for 5 minutes.

Each PCR product was digested with BglII (TAKARA), separated and purified by agarose gel, and cloned into the BglII or BamHI site of V913 (vector name: VH 21-12). 3' HPRT-loxP the loxP sequence for oligo synthesis was cloned into XbaI site of V820(Lexicon genetics). The exons from No. 3 to No. 9 of the HPRT gene, 3' HPRT-loxP, were cloned into EcoRI and AscI (vector name: X3.1) of V907(Lexicon genetics). Further, the PGKneo sequence excised with KpnI and NotI was cloned into KpnI site of X3.1 and EcoRI (vector name: X4.1). PGKneo-loxP-3' HPRT excised from X4.1 using KpnI and AscI was cloned into KpnI site and AscI site of V913 (vector name: pVNLH). The smoothed HS4-CAG-EGFP-HS4 (gifted by doctor Bao of the university of Osaka and doctor Felsenfeld of NIH) was cloned into the EcoRV site of pVNLH after digestion with NotI and SalI (vector name: pVGNLH). The GFP-PGKneo-loxP-3' HPRT cassette excised from pVGNLH using SalI and AscI was cloned into the XhoI site and AscI site of VH21-12 (vector name: pMAC 1). The targeting vector, the target sequence and the chromosomal allele produced by homologous recombination are shown in FIG. 11.

[ A.2] transfection and isolation of G418-resistant clones

The culture of chicken DT40 cells was performed in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco, hereinafter, FBS), 1% chicken serum (Gibco), and 10-4M 2-mercaptoethanol (Sigma). About 10 of DT40(MAC) -1⁷The cells were washed once with the non-supplemented RPMI1640 medium, suspended in 0.5ml of the non-supplemented RPMI1640 medium, added with 25. mu.g of the targeting vector pMAC1 linearized with the restriction enzyme NotI (TAKARA), transferred into a Cuvette for electroporation (Cuvette) (BioRad), and allowed to stand at room temperature for 10 minutes. The cuvette was set in a gene pulser (BioRad), and a voltage was applied under the conditions of 550V and 25. mu.F. After standing at room temperature for 10 minutes, the cells were cultured for 24 hours. The medium containing G418(1.5mg/ml) was replaced, and the mixture was injected into 2 96-well culture plates and subjected to selective culture for about 2 weeks. A total of 14 resistant colonies obtained by 2 transfections were separated and propagated for further analysis (clone name: DT40(MAC1))

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

In order to extract genomic DNA of the G418-resistant strain and select recombinants as templates, PCR was performed using the following primers to confirm whether recombination occurred site-specifically on mouse chromosome 11. The primer sequences are shown below.

kj neo: 5'-CATCGCCTTCTATCGCCTTCTTGACG-3' (Serial number 9)

m117R (supra)

m115L (above)

EGFP-F (L) 5'-CCTGAAGTTCATCTGCACCA-3' (SEQ ID NO. 10)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer was used as a thermal cycler, LA Taq (TAKARA) was used as Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 35 cycles of heat denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 2 out of 88 clones were positive in all primer sets, and therefore, the following analysis was performed using the 2 clones.

TABLE 5

[ A.3.2] two-color FISH analysis

Two-color FISH analysis was performed on DT40(MAC1) -52 and DT40(MAC1) -58 obtained as described above according to Sonogen et al (FISH protocol, Xiu run Co., 1994). FISH analysis was performed using mouse cot-1DNA and GFP-PGKneo-loxP-3' HPRT cassette as probes, and FITC signal derived from the probes was detected in the vicinity of centromere of mouse chromosome 11 fragment targeted with loxP sequence, and a signal not present in mouse chromosome 11 fragment before targeting (for example, DT40(MAC) -1) was detected as a negative control, thereby visually confirming that site-specific recombination was caused (FIG. 12). From these results, the following conclusions can be drawn: DT40 cell clones carrying the mouse artificial chromosome vector MAC1 were obtained.

TABLE 6

[B] Introduction of MAC1 into CHO cells from DT40 cells containing mouse artificial chromosome vector MAC1

Mouse artificial chromosome vector MAC1 is introduced into CHO cells in order to introduce mouse artificial chromosome vector MAC1 into mouse ES cells via CHO cells, or stably insert a target gene (group) or the like into CHO cells via loxP, which is a DNA sequence insertion site of mouse artificial chromosome vector MAC1, for example, CYP3A cluster or the like.

[ B.1] isolation of micronuclear cell fusion and drug-resistant clones

CHOhprt-deficient cells (obtained from the Japanese research resource repository (ヒユ - マンサイエンス research resources バンク) under accession number JCRB0218), namely CHO (HPRT), were treated in the same manner as described above using DT40(MAC1)52 and 58 as recipient cells^-) The micronucleus cell fusion method was performed. A total of 24 resistant colonies obtained by 2 micronuclear cell fusions were separated and proliferated, and then analyzed (clone name: CHO (HPRT)^-；MAC1))

[ B.2] screening of drug-resistant clones

[ B.2.1] PCR analysis

Genomic DNA of the G418-resistant strain was extracted as a template, and recombinants were selected, and PCR was performed using the following primers to confirm whether mouse artificial chromosome MAC1 could be introduced into CHO cells. The primer sequences are shown below.

kj neo (above)

m117R (supra)

m115L (above)

EGFP F (L) (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 20 out of 24 clones were positive in all the primer sets, and the 20 clones were used for subsequent analyses.

TABLE 7

[ B.2.2] Monochromatic FISH analysis

For the CHO (HPRT) obtained above^-(ii) a MAC1), reported by Shinohara et al (human molecular Genetics, 10: 1163-1175, 2001) was subjected to FISH analysis using mouse Cot-1DNA as a probe, and it was confirmed that the mouse artificial chromosome vector MAC1 was introduced into CHO cells at a ratio of 95% in 5 out of 20 clones (FIG. 13).

TABLE 8

From the above results, the following conclusions can be drawn: the mouse artificial chromosome vector MAC1 can be introduced into CHO cells.

[C] Introduction of mouse Artificial chromosome vector MAC1 into mouse ES cells from CHO cells containing mouse Artificial chromosome vector MAC1

In order to examine the stability of the mouse ES cells and the mouse artificial chromosome vector MAC1 in mouse individuals, a chimeric mouse and a progeny transmission mouse containing the mouse artificial chromosome vector MAC1 were prepared by introducing the mouse artificial chromosome MAC1 into the mouse ES cells.

[ C.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) to be used as recipient cell^-(ii) a MAC1) -3, 5, 8, and 22 were cultured in a cell culture dish, and at the time of reaching confluence, the culture was changed to F12 medium supplemented with 20% FBS and 0.1. mu.g/ml autumn acrylamide, and after culturing for 48 hours, the culture was changed to F12 medium supplemented with 20% FBS and 0.1. mu.g/ml autumn acrylamide, and then the cells were cultured overnight to form minicells. The culture solution was removed, and cytochalasin B (10. mu.g/ml, Sigma) solution previously incubated at 37 ℃ was filled in a flask for centrifugation, and centrifugation was performed at 8000rpm at 34 ℃ for 1 hour. Minicells were suspended in serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, the resulting mixture was centrifuged at 2000rpm for 10 minutesHeart suspended in 5ml serum-free DMEM medium. Minicells were suspended in 5ml serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, centrifugation was carried out at 2000rpm for 10 minutes.

As donor cells, B6 (HPRT) which was a 6 TG-treated HPRT-deficient strain obtained by treating B6-ES and B6-ES cells which were ES cells of a C57B6 system mouse was used^-) TT2F, which is ES cells of C57B6xCBA system F1 mice, and HPRT-deficient strain KO56 (HPRT) in which 6TG treatment was performed on TT2F cells (HPRT system)^-) For the culture, 10% FCS, LIF (Muerin Leukemia inhibition Factor), 1 × 10% were added to DMEM (Dulbecco's modified eagle's Medium-high glucose: SIGMA)^-5M2-ME (2-mercaptoethanol: SIGMA), L-glutamine (3.5 g/ml: GIBCO), sodium pyruvate solution (3.5 g/ml: GIBCO), MEM Nonessarial amino acid (0.125 mM: GIBCO) in 5% CO₂The culture was carried out at 37 ℃. Mouse ES cells were washed 2 times with PBS (-) on the cell surface, then the cells were dispersed by trypsin treatment, recovered in a culture medium supplemented with 10% FBS in DMEM medium, centrifuged at 1500rpm, the supernatant was removed, resuspended in 5ml of serum-free medium, gently added to the serum-free medium containing the centrifuged particles of minicells, and centrifuged at 1200 rpm. Removing supernatant, dissolving PEG1000(Wako) solution [ 5g of PEG1000 completely dissolved in serum-free DMEM medium, adding 1ml of dimethyl sulfoxide, and filtering for sterilization]Fusion was correctly performed with 0.5ml for 1 min 30 sec. 13ml of serum-free medium (DMEM) was gently added and centrifuged at 1200 rpm. The supernatant was removed, and a normal culture solution of mouse ES cells was inoculated into 2 cell culture dishes of 10cm in diameter and cultured overnight using mitomycin-treated G418-resistant mouse embryo fibroblasts as feeder cells. G418 was added to 250. mu.g/ml, and selection culture was performed for 3 to 4 weeks (clone name: B6-ES (MAC1) and B6 (HPRT)^-(ii) a MAC1) and KO56 (HPRT)^-(ii) a MAC 1)). For B6-ES (MAC1) and B6 (HPRT)^-(ii) a MAC1) and KO56 (HPRT)^-(ii) a MAC1), a total of 32 resistant colonies obtained by two micronuclear cell fusions were separated and proliferated, and the following analysis was performed. For TT2F (MAC1), and 30 resistant colonies in total obtained by 4 times of micronucleus fusion were separated and proliferated, and analyzed after FISH analysis.

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of the G418-resistant strain as a template for selection of recombinants, PCR was performed using the following primers to confirm whether mouse artificial chromosome MAC1 could be introduced into mouse ES cells. The primer sequences are shown below.

m115L (above)

EGFP F (L) (supra)

kj neo (above)

m117R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 10 minutes were carried out. As a result of PCR, 30 out of 32 clones were positive in all the primer sets, and 14 clones were used for the subsequent analysis.

TABLE 9

[ C.2.2] Monochromatic FISH analysis

For the above obtained B6-ES (MAC1) and B6 (HPRT)^-(ii) a MAC1) and KO56 (HPRT)^-(ii) a MAC1), reported by Shinohara et al (Human Molecular Genetics, 10: 1163-1175, 2001) was performed by FISH analysis using mouse secondary satellite DNA as a probe, and it was confirmed that 85% of all 14 clones were presentThe above ratio MAC1 was introduced into mouse ES cells. In addition, the number of endogenous mouse chromosomes confirmed as a normal karyotype was 40 in the case of B6-ES or 39 in the case of KO 56. At B6 (HPRT)^-(ii) a MAC1), 40 karyotype clones could not be obtained. Similarly, with respect to TT2F (MAC1), 7 clones were analyzed, and it was confirmed that 90% or more of all the 7 clones were introduced. In addition, for 3 clones out of 7 clones, the number of endogenous mouse chromosomes confirmed as normal karyotypes was 39.

From the above results, the following conclusions can be drawn: mouse artificial chromosome vector MAC1 can be introduced into mouse ES cells (fig. 14).

Watch 10

EXAMPLE 3 construction of mouse Artificial chromosome vector CYP3A-MAC

For the mouse artificial chromosome vector MAC1, the CYP3A cluster, which is a gene group of human drug metabolizing enzymes, was transposable cloned using the Cre/loxP system to construct CYP 3A-MAC. In addition, stability of CYP3A-MAC in mouse ES cells was examined, and progeny transmission mice into which CYP3A-MAC was introduced were prepared, to examine stability in individual tissues. In addition, in the offspring-transmitted mice, the tissue-specific gene expression of CYP3A gene was examined (fig. 2).

[A] Site-specific insertion of loxP site into AC004922 of human chromosome 7

In order to transpose and insert the loxP sequence into the mouse artificial chromosome vector MAC1 via the loxP sequence, the loxP sequence was inserted into DT40 cells into AC004922 proximal to the CYP3A gene cluster of human chromosome 7 (hChr 7).

[ A.1] preparation of targeting vector pMPLoxyphyll

A targeting vector pMPloxPHyg for inserting a recognition sequence loxP of Cre recombinase into the region AC004922 located near the CYP3A locus on human chromosome 7 and on the centromeric side (about 300Kb on the centromeric side) was prepared as follows. First, the AC004922 genomic region was amplified by PCR using the following primers.

p450loxP 7L; 5'-GGCCTAGAGCCTGGACTCATTCATTCAA-3' (Serial number 11)

p450loxP 7R; 5'-GACAGATGTCATGCCCCAGGTAGGTATG-3' (Serial number 12)

The basic plasmid for inserting the loxP site was V901(Lexicon genetics). For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (Takara Shuzo) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation at 98 ℃ for 20 seconds and 68 ℃ for 7 minutes were carried out. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400(Clontech (クロ - ンテツク)). Then, it was cleaved with restriction enzymes BamHI (Boringer) and EcoRI (ニツポンジ - ン) and BglII (ニツポンジ - ン) and gel-filtered with CHROMASPIN-TE1000 (Clontech). The PCR fragments (3.7kb and 3.0kb) were cloned into EcoRI and BamHI or BglII sites of the V901 plasmid (vector name: V901-NP 21). Next, V901-NP21 was cleaved with restriction enzymes AscI (NEB) and KpnI, and a DNA fragment containing loxP was excised from the cassette vector 5' HPRT-loxP-Hyg-TK (Kazuki et al, Gene Therapy: PMID: 21085194, 2010) with restriction enzymes AscI and KpnI, followed by ligation. A vector having the loxP site in the same orientation as the cloned AC004922 genomic fragment was used as the targeting vector pMPluxPHyg. The size of the final loxP insert construct is 12 kb. Targeting vectors, target sequences and chromosomal alleles produced by homologous recombination are shown (FIG. 15 a).

[ A.2] transfection and isolation of drug-resistant clones

The targeting vector pMPloxPHyg prepared above was linearized with the restriction enzyme NotI (TAKARA) in the same manner as described above, transfected into chicken DT40 cells (clone DF141) carrying the human chromosome 7 fragment (site-specifically cleaved at AF 006752) prepared by the method described in WO01/011951, replaced with a medium containing hygromycin B (1.5mg/ml), and individually injected into 3 96-well culture plates for about 2 weeks of selective culture. A total of 96 resistant colonies obtained by 5 transfections were separated and propagated, followed by analysis (clone name: DT40(hCHr 7-loxP)).

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

Genomic DNA was extracted from the hygromycin-resistant clones using the Puregene DNA Isolation Kit (Gentra System Co.), and homologous recombinants were identified by PCR using the following set of 2 primers.

Identification of homologous recombinants was performed by PCR using the following 2 sets of primers.

p450loxP 14L; 5'-AGTTCTTTTGAGGGCCTAGAGCCTGGAC-3' (Serial number 13)

p450loxP 14R; 5'-AAAGGACAGAAGGAGGGAGCAACAGGAT-3' (Serial number 14)

p450loxP 16L; 5'-TCTGGGCATCAGTGTCCTCTCCAGTAAA-3' (Serial number 15)

p450loxP 16R; 5'-TTGGCGACATCCAATGCTAGTGCTATTC-3' (Serial number 16)

For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 4 minutes at 68 ℃ were carried out. As a result of screening 96 clones, 36 clones were identified as homologous recombinants.

[ A.3.2] southern blot analysis

The 6 clones whose recombination was confirmed by the above PCR analysis were subjected to southern blot analysis as follows. The genomic DNA is subjected toGeneScreen plus hybridization transfer membranes (NENTM Life Science Products, Inc.) were subjected to alkali blotting by treatment with restriction enzyme EcoRI (TAKARA) and electrophoresis in 0.8% agarose gel. For this filter, DNA hybridization was performed using an MPp probe amplified by PCR for the gene sequence in AC004922 and identification of homologous recombinants was performed. The MPp probe was prepared as follows: PCR was carried out using the following primers and DF141 genomic DNA as a template, and random priming was performed using the PCR product as a template³²p-labeled DNA probes (Amersham, according to the protocol noted).

Primers for MPp Probe preparation:

MPp 6L; 5'-TGGAGACGTTGTTTAGCCTCTCCTCCTC-3' (Serial number 17)

MPp 6R; 5'-CACAGCTTAGAGGCCATTCCCATAGTCC-3' (Serial number 18)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, EXTAQ (TAKARA) is used as a thermal cycler, and EXTAQ and dNTP (dATP, dCTP, dGTP, dTTP) are used according to the recommended conditions for labeling. The temperature and cycle conditions were 35 cycles of 1 cycle of 93 ℃ for 1 minute, 54 ℃ for 1 minute and 72 ℃ for 1 minute after 5 minutes of thermal denaturation at 93 ℃. By DNA hybridization, it was predicted that a band of about 10.9kb was detected in non-homologous recombinants and about 8.9kb was detected in homologous recombinants (FIG. 15 b). As a result of DNA hybridization, all of the 6 clones were targeted homologous recombinants.

[ A.3.3] two-color FISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of 6 of the above clones confirmed to be recombined using human cot-1DNA and hygromycin as probes, human chromosome 7 was not transposed to the host chromosome in all the clones, and a hygromycin-derived signal was detected in the vicinity of 7q22, thereby confirming that recombination was site-specifically caused. From the above results, the following conclusions can be drawn: in the human chromosome 7 fragment, a loxP site as a gene introduction site is site-specifically inserted.

[B] Site-specific cleavage in human chromosome 7 region AC073842 in hCHR7-loxP

As in WO2009/063722(PCT/JP2008/068928), in order to delete a gene of a mouse individual strongly related to generation of the CYP3A gene cluster distal to human chromosome 7, site-specific chromosome deletion, i.e., telomere truncation, was performed.

[ B.1] preparation of targeting vector pTELhisD-PT

A targeting vector pTELhisD-PT for inserting a human telomere sequence into the AC073842 region located near the CYP3A locus on human chromosome 7 and on the telomere side (approximately 150Kb telomere side) was prepared as follows. First, the AC073842 genomic region was amplified by PCR using the following primers.

PT 1L; 5'-TGCGGTGAAGGTCCAAGGAGATAGATTT-3' (Serial number 19)

PT 2R; 5'-TCTAGCAGAGAGATGGTGGCAGGATTCA-3' (Serial number 20)

For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400 (Clontech). Then, the cells were digested with restriction enzymes BamHI (Boringer) and BglII (ニツポンジ - ン), and gel-filtered with CHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the BamHI site of the plasmid pTELhisD (Kuroiwa et al, Nature Biotech., 20: 88, 2002). The AC073842 genomic sequence was oriented telomere → centromere, and thus, the vector in which the cloned AC073842 genomic fragment was oriented with the human telomere sequence was used as the targeting vector ptelhis d-PT. The size of the final long-armed site-specific cleavage construct was 14.4 kb. Targeting vectors, target sequences and chromosomal alleles produced by homologous recombination are shown (FIG. 16).

[ B.2] transfection and isolation of histidinol-resistant clones

The targeting vector pTELhisD-PT prepared above was linearized with restriction enzyme SrfI (Toyobo Co., Ltd.) in the same manner as described above, transfected into the clone DT40(hCHr7-loxP)122 prepared above, and replaced with a medium containing histidinol (0.5mg/ml), and the cells were individually plated on 10 96-well plates and selectively cultured for about 2 weeks. A total of 335 resistant colonies obtained by 5 transfections were separated and propagated for subsequent analysis (clone name: DT40(hCHr 7-loxP-tel)).

[ B.3] screening of homologous recombinants

[ B.3.1] PCR analysis

In order to select recombinants using genomic DNA of a histidinol-tolerant strain as a template, PCR was performed using primers located at the telomere side of the cleavage site or less as a primary selection to confirm whether site-specific cleavage occurred. The primer sequences are shown below.

COPS 6-1L; 5'-TGAGGGTACTTGAAGGGCTGATG-3' (Serial number 21)

COPS 6-1R; 5'-CAGGGGCTGCTCCCCTTTTATTA-3' (Serial number 22)

AP4M 1-1L: 5'-CCTAACATCGTGTCCCAGCTCA-3' (Serial number 23)

AP4M 1-1R: 5'-TCCTTTCAGACCCCTTCATCTTAG-3' (Serial number 24)

LRCH 4-2L: 5'-TTCAGCCCCAACCAAAGACACTA-3' (Serial number 25)

LRCH 4-1R: 5'-GCCCCGAACCCCTACAAATATAGA-3' (Serial number 26)

STAG 3-1L: 5'-GGGCCTCCAATAAGTGTCCCATA-3' (Serial number 27)

STAG 3-1R: 5'-TTGCTGACTTAGTTGCAGCAGGA-3' (Serial number 28)

PILRB-2L: 5'-CCCATTGGCAAGATACATGGAGA-3' (Serial number 29)

PILRB-2R: 5'-AGTGTGGATGCTCCTGGATGAAG-3' (Serial number 30)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer was used as a thermal cycler, Ampli Taq Gold (Applied Biosystems) was used as a Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 95 ℃ for 10 minutes, followed by 30 cycles at 95 ℃ for 20 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds. As a result of PCR, 2 clones out of 433 clones were positive.

Next, 2 clones out of 433 clones detected by the above primers were confirmed to have caused site-specific homologous recombination by PCR using the following primers. The sequence is as follows.

PT 2R; (above-mentioned)

hisD 2: 5'-GTAAACGCCCTCAAGGAGCAAGCATGA-3' (Serial number 31)

hisD 3: 5'-TGTGACCAAAGATTTAGCGCAGTGCGT-3' (Serial number 32)

Using the above primers, LATaq (Takara Shuzo) was used for PCR, and buffers and dNTPs (dATP, dCTP, dGTP and dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. A band of about 8kb was detected only in2 clones which site-specifically caused recombination. No band was detected in negative controls DT40, DT40(hCHR 7-loxP).

TABLE 11

[ B.3.2] two-color FISH analysis

FISH analysis was according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of 2 clones among the above clones confirmed to be recombined using human cot-1DNA and histidinol as probes, human chromosome 7 into which the loxP sequence was inserted was not transposed to the host chromosome in all the clones, and a signal derived from histidinol was detected at the end of the human chromosome 7 fragment, thereby confirming that recombination was caused site-specifically.

From the above results, the following conclusions can be drawn: in clones DT40(hCHR7-loxP-tel)608 and 748, the cleavage was carried out at the distal end of AC073842 which is more telomeric than the CYP3A gene cluster region.

TABLE 12

[C] hCHr7-loxP-tel introduction from DT40 containing hCHr7-loxP-tel into CHO cells containing MAC 1.

In order to transpose the human CYP3A gene cluster region into the mouse artificial chromosome vector MAC1 via the loxP sequence in CHO cells, hCHR7-loxP-tel was introduced into CHO cells containing the mouse artificial chromosome vector MAC 1.

[ C.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) that is CHOhprt deficient cells (obtained from the Japan health science research resource Bank, accession number JCRB0218) containing MAC1 was treated with DT40 (hCHhr 7-loxP-tel)608 and 748 as recipient cells in the same manner as described above^-(ii) a MAC1) was subjected to the micronucleus fusion method. A total of 48 resistant colonies obtained by 5 micronuclear cell fusions were separated and proliferated, and the following analysis was performed (clone name: CHO (HPRT)^-；MAC1，hChr7-loxP-tel))。

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of hygromycin-resistant strains as a template for selection of recombinants, PCR was performed using the following primers, and it was confirmed that human chromosome 7 fragment was introduced into CHO cells containing MAC 1. The primer sequences are shown below.

m115L (above)

EGFP (F) L (above)

kj neo (above)

m 116R: 5'-CCCAGGAATCAGTCAGGAAGGCTGTAA-3' (Serial number 33)

P450loxP 14L: (above-mentioned)

Hyg F (244): 5'-GAATTCAGCGAGAGCCTGAC-3' (Serial number 34)

Hyg R (696): 5'-GATGTTGGCGACCTCGTATT-3' (Serial number 35)

P450loxP 16R: (above-mentioned)

CYP3a 4R: 5'-GGCTGCATCAGCATCATCTA-3' (Serial number 36)

CYP3a 4F: 5'-GCAAGACTGTGAGCCAGTGA-3' (Serial number 37)

CYP3a 5R: 5'-TCAGCTGTGTGCTGTTGTTTGC-3' (Serial number 38)

CYP3a 5F: 5'-ATAGAAGGGTCTGTCTGGCTGG-3' (Serial number 39)

CYP3a 7R: 5'-GAGTTAATGGTGCTAACTGGGG-3' (Serial number 40)

CYP3a 7F: 5'-ACCCTGAAATGAAGACGGGC-3' (Serial number 41)

3A 44L: 5'-TCCCCCTGAAATTAAGCTTA-3' (Serial number 42)

3A 43R: 5'-TGAGGTCTCTGGTGTTCTCA-3' (Serial number 43)

3A 73L: 5'-TCCCCCTGAAATTACGCTTT-3' (Serial number 44)

3A 73R: 5'-CATTTCAGGGTTCTATTTGT-3' (Serial number 45)

PT 2R: (above-mentioned)

hisD 1: 5'-GTATTGGTCACCACGGCCGAGTTTCCGC-3' (Serial number 46)

hisD 2: (above-mentioned)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 19 out of 48 clones were positive in all the primer sets, and 20 clones containing 1 negative clone were used for the subsequent analysis.

TABLE 13A

TABLE 13B

[ C.2.2] two-color FISH analysis

The CHO (HPRT) obtained above was subjected to the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC1, hCHr7-loxP-tel) was subjected to FISH analysis using mouse Cot-1DNA and human Cot-1DNA as probes, and as a result, it was confirmed that MAC1 and hCHr7-loxP-tel were introduced into CHO cells at 1 copy or 2 copies in 18 clones other than the above-mentioned 1 negative clone (FIG. 17).

TABLE 14

From the above results, the following conclusions can be drawn: hCHr7-loxP-tel can be introduced into CHO cells containing mouse artificial chromosome vector MAC 1.

[D]CHO(HPRT^-(ii) a MAC1, hCHr7-loxP-tel) clone in the region surrounding the human CYP3A gene cluster (AC 004922-human CYP3A gene cluster-AC 073842)1Mb site-specific transposition into the MAC1 vector

In order to stably maintain a1 Mb-sized DNA, i.e., the human CYP3A gene cluster, in a mouse individual, a transposition was inserted into the mouse artificial chromosome vector MAC1 (fig. 18).

[ D.1] transfection and isolation of HAT-resistant clones

The CHO (HPRT) obtained above was subjected to lipofection^-(ii) a MAC1, hCHr7-loxP-tel) -6, 9, 12, and 47 were introduced, and Cre 3. mu.g was introduced into 6-well (well) cells that reached 90% confluence according to the commercially available protocol (Invitrogen). In 2-week culture under HAT selection culture, resistant colonies appeared, and a total of 42 colonies obtained by 4 introductions were separated and proliferated for subsequent analysis (clone name: CHO (CYP3A-MAC1, hCHR 7-. DELTA.CYP 3A)).

[ D.2] screening of drug-resistant clones

[ D.2.1] PCR analysis

In order to extract genomic DNA of the HAT-tolerant strain as a template and screen reciprocal transposition clones, PCR was performed using the following primers to confirm whether chromosomal reciprocal transposition was caused on the human chromosome 7 fragment and MAC 1. The primer sequences are shown below.

P450loxP16R (above)

Hyg R (696) (above)

kj neo (above)

P450loxP14L (above)

m115L (above)

m 116R (supra)

CYP3A 4R (supra)

CYP3A 4F (supra)

CYP3A 5R (supra)

CYP3A 5F (supra)

CYP3A 7R (supra)

CYP3A 7F (supra)

3A 44L (supra)

3A 43R (supra)

3A 73L (supra)

3A 73R (supra)

TRANS L1: 5'-TGGAGGCCATAAACAAGAAGAC-3' (Serial number 47)

TRANS R1: 5'-CCCCTTGACCCAGAAATTCCA-3' (Serial number 48)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 27 out of 42 clones were positive in all the primer sets, and the 27 clones were used for subsequent analyses.

Watch 15

[ D.2.2] two-color FISH analysis

FISH analysis using mouse Cot-1DNA and Human Cot-1DNA as probes was performed on 27 clones of CHO (CYP3A-MAC1, hCHR 7-. DELTA.CYP 3A) obtained as described above by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001), and as a result, signals due to Human chromosome 7 were observed on MAC1 composed of a mouse chromosome 11 fragment containing a loxP sequence at a ratio of 50% or more among 25 clones out of the 27 clones (FIG. 19).

TABLE 16

From the above results, the following conclusions can be drawn: the CYP3A cluster on the human chromosome 7 fragment with the loxP sequence inserted, which is 1Mb cloned by transposition to each other, can be used on the mouse artificial chromosome vector MAC 1.

EXAMPLE 4 construction of mouse Artificial chromosome vector MAC2

Mouse artificial chromosome vector MAC2 (FIG. 3) was constructed in which 5' HPRT-loxP-PGKhyg type loxP site was inserted as a DNA insertion sequence into mouse artificial chromosome vector MAC. The 5' HPRT-loxP-PGKhyg type loxP site was inserted into the HAC vector 21HAC2 derived from chromosome 21 described in Kazuki et al report (Gene therapy: PMID: 21085194, 2010), and the expression of HAC and MAC genes were compared using the same vector. The gene transfer vector for insertion into 21HAC2 can be used as it is without a step of vector preparation.

[A] Insertion of 5' HPRT-loxP-PGKhyg type loxP sequence into mouse artificial chromosome MAC

[ A.1] preparation of 5' HPRT-loxP-PGKhyg type loxP targeting vector

The basic plasmid for loxP site insertion was VH21-12 prepared as described above. The 5' HPRT-loxP-PGKhygro cassette excised from the above X6.1 by KpnI and AscI was cloned into KpnI and AscI sites of V907(Lexicon genetics) (vector name: pV 907-AML). Furthermore, the 5' HPRT-loxP-PGKhygro cassette was excised from pV907-AML using XhoI and SalI and cloned into the XhoI site of VH21-12 (vector name: pMAC 2). The targeting vector, the target sequence, and the chromosomal allele produced by homologous recombination are shown in FIG. 20.

[ A.2] transfection and isolation of drug-resistant clones

The targeting vector pMAC2 prepared above was linearized with the restriction enzyme NotI (TAKARA) in the same manner as described above, transfected into the clone DT40(MAC) prepared above, replaced with a hygromycin (1.5mg/ml) containing medium, and injected into 2 96-well plates for about 2 weeks of selective culture. A total of 45 resistant colonies obtained by 1 transfection were separated and propagated for subsequent analysis (clone name: DT40(MAC 2)).

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

In order to extract genomic DNA of hygromycin-resistant strains as templates and select recombinants, PCR was performed using the following primers to confirm whether recombination occurred site-specifically at the MAC site in the mouse artificial chromosome vector. The primer sequences are shown below.

TRANS-L (above)

m 116R (supra)

m117R (supra)

m 114L: 5'-ACTCCTAAGGGAGTTGGTGCTGTTGGTG-3' (Serial number 49)

m115L (above)

hygF (244): (above-mentioned)

hygR (696): (above-mentioned)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 7 minutes at 68 ℃ were carried out. As a result of PCR, 8 clones out of 45 clones were positive in all primer sets, and therefore, 6 clones randomly selected from the 8 clones were used for the subsequent analysis.

TABLE 17

[ A.3.2] two-color FISH analysis

Two-color FISH analysis was performed on 6 clones of DT40(MAC2) obtained as described above according to Songen et al (FISH protocol, Xiu run Co., 1994). In contrast to FISH analysis using mouse cot-1DNA and 5' HPRT-loxP-PGKhygro cassette as probes, which showed that the rate of signal derived from the probe was significantly 10% in the mouse artificial chromosome vector MAC, which is the mouse chromosome 11 fragment before targeting of the negative control, the signal derived from the probe was detected in 50% or more of the 6 clones of DT40(MAC2), and thus site-specifically induced recombination was visually confirmed in the 6 clones (fig. 21). From these results, the following conclusions can be drawn: DT40 cell clones carrying the mouse artificial chromosome vector MAC2 were obtained.

Watch 18

[B] Introduction of MAC2 into CHO cells from chicken DT40 cells containing mouse artificial chromosome vector MAC2

In order to stably introduce mouse artificial chromosome vector MAC2 into mouse ES cells, the vector was introduced into CHO cells. In addition, mouse artificial chromosome vector MAC2 was introduced into CHO cells in order to stably insert a target gene or the like (for example, GFP gene or the like) via loxP, which is the DNA sequence insertion site of mouse artificial chromosome vector MAC 2.

[ B.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) that is a CHOhprt deficient cell (obtained from the Japan health science research resource Bank, accession number JCRB0218) was used as described above using DT40(MAC2) -5 and 17 as recipient cells^-) The micronucleus cell fusion method was performed. A total of 44 resistant colonies obtained by 2 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: CHO (HPRT)^-；MAC2))。

[ B.2] screening of drug-resistant clones

[ B.2.1] PCR analysis

In order to extract genomic DNA of the hygromycin-resistant strain as a template for selection of recombinants, PCR was performed using the following primers to confirm whether the mouse artificial chromosome vector MAC2 could be introduced into CHO cells. The primer sequences are shown below.

TRANS-L (above)

m 116R (supra)

m117R (supra)

m 114L (above)

m115L (above)

hygF (244): (above-mentioned)

hygR (696): (above)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 14 out of 44 clones were positive in all the primer sets, and from the 14 clones, random selection was performed for subsequent analysis.

Watch 19

[ B.2.2] two-color FISH analysis

By a method according to Songen et al (FISH protocol, Xiu Run Co., 1994) on CHO (HPRT) randomly selected from the above^-(ii) a MAC2) was subjected to FISH analysis using mouse Cot-1DNA and 5' HPRT-loxP-PGKhygro cassette as probes, and it was confirmed that MAC2 was introduced into CHO cells at a rate of 95% in 8 out of 9 clones (fig. 22).

Watch 20

From the above results, the following conclusions can be drawn: mouse artificial chromosome vector MAC2, in which a loxP sequence is inserted as a gene insertion site into mouse artificial chromosome MAC, which is a chromosome fragment derived from mouse chromosome 11, can be introduced into CHO cells.

[C] Introduction of mouse Artificial chromosome vector MAC2 into mouse ES cells from CHO cells containing mouse Artificial chromosome vector MAC2

[ C.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) to be used as recipient cell^-(ii) a MAC2) -13, 18 were cultured on cell culture dishes until reaching sinkAt the time of completion, the medium was replaced with F12 medium supplemented with 20% FBS and 0.1. mu.g/ml autumn acrylamide, and cultured for another 48 hours, and then replaced with F12 medium supplemented with 20% FBS and 0.1. mu.g/ml autumn acrylamide, and then cultured overnight to form minicells. The culture solution was removed, and cytochalasin B (10. mu.g/ml, Sigma) solution previously incubated at 37 ℃ was filled in a flask for centrifugation, and centrifugation was performed at 8000rpm at 34 ℃ for 1 hour. The micronucleus cells (also referred to as "minicells") were suspended in serum-free DMEM medium and purified using 8 μm, 5 μm, and 3 μm filters. After purification, the suspension was centrifuged at 2000rpm for 10 minutes and suspended in 5ml of serum-free DMEM medium. Minicells were suspended in 5ml serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, centrifugation was carried out at 2000rpm for 10 minutes.

As donor cells, B6-ES, which was an ES cell of a C57B6 system mouse obtained from Japanese Clea, and B6 (HPRT) which was an HPRT-deficient strain obtained by subjecting the ES cell to 6TG treatment, were used^-) And KO56 (HPRT) which is an HPRT-deficient strain of TT2F cells^-) For the culture, 10% FCS, LIF (Muerin Leukemia inhibition Factor), 1 × 10% were added to DMEM (Dulbecco's Modified Eagle's Medium-high glucose: SIGMA)^-5M2-ME (2-mercaptoethanol: SIGMA), L-glutamine (3.5 g/ml: GIBCO), sodium pyruvate solution (3.5 g/ml: GIBCO), MEM non-essential amino acids (0.125 mM: GIBCO) in 5% CO₂The culture was carried out at 37 ℃. Mouse ES cells were washed 2 times with PBS (-), then the cells were dispersed by trypsin treatment, recovered in a culture medium supplemented with 10% FBS in DMEM medium, centrifuged at 1500rpm, the supernatant removed, resuspended in 5ml serum-free medium, gently added to serum-free medium containing the centrifuged particles of minicells, and centrifuged at 1200 rpm. Removing supernatant, dissolving PEG1000(Wako) solution [ 5g of PEG1000 completely dissolved in serum-free DMEM medium, adding 1ml of dimethyl sulfoxide, and filtering for sterilization]Fusion was correctly performed with 0.5ml for 1 min 30 sec. 13ml of serum-free medium (DMEM) was gently added thereto, and centrifugation was performed at 1200 rpm. The supernatant was removed, and a normal culture medium of mouse ES cells was added thereto, followed by mitomycin-treated G418 resistant mouse embryo fibroblasts were used as feeder cells, inoculated into 2 cell culture dishes of 10cm in diameter, and cultured overnight. Hygromycin was added to the cells at a concentration of 250. mu.g/ml, and the cells were selectively cultured for 3 to 4 weeks. A total of 28 resistant colonies obtained by 2 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: B6-ES (MAC2) and B6 (HPRT)^-(ii) a MAC2) and KO56 (HPRT)^-；MAC2))。

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of the hygromycin-resistant strain as a template for selection of recombinants, PCR was performed using the following primers to confirm whether mouse artificial chromosome MAC2 could be introduced into mouse ES cells. The primer sequences are shown below.

TRANS L (above)

m 116R (supra)

m 114L (above)

hyg R (696) (above)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 10 minutes were carried out. As a result of PCR, 27 out of 28 clones were positive in all the primer sets, and the subsequent analysis was performed with 3 clones randomly selected from the 27 clones.

TABLE 21

[ C.2.2] Monochromatic FISH analysis

The clones of the mouse ES (MAC2) obtained as described above were subjected to FISH analysis using mouse secondary satellite DNA as a probe by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001), and it was confirmed that MAC2 was introduced into mouse ES cells at a rate of 80% or more in 1 of the 3 clones and that the number of endogenous mouse chromosomes of the normal karyotype as KO56 cells was 39.

From the above results, the following conclusions can be drawn: mouse artificial chromosome vector MAC2, in which a loxP sequence as a gene insertion site is inserted into mouse artificial chromosome MAC, which is a chromosome fragment derived from mouse chromosome 11, can be introduced into mouse ES cells (fig. 23).

TABLE 22

[D] The stability in vitro can be tested using mouse ES cells carrying the mouse artificial chromosome vector MAC2 as described in example 8. Furthermore, chimeric mice were prepared from the ES cells, and mouse system TC (MAC2) in which MAC2 was transmitted as progeny was prepared. In addition, using the TC (MAC2) mouse system described above, the stability of MAC2 in somatic cells can be examined.

EXAMPLE 5 construction of mouse Artificial chromosome vector MAC3

Mouse artificial chromosome vector MAC3 (FIG. 4) was constructed in which a PGKneo-loxP-3' HPRT type loxP site was inserted as a DNA insertion sequence into mouse artificial chromosome MAC. Stability of mouse artificial chromosome vector MAC3 in mouse ES cells was examined, and further stability in individual tissues was examined by making progeny transmission mice into which MAC3 was introduced.

[A] Insertion of loxP site of PGKneo-loxP-3' HPRT type into mouse artificial chromosome MAC

[ A.1] preparation of PGKneo-loxP-3' HPRT type loxP targeting vector

The basic plasmid for loxP site insertion was VH21-12 prepared as described above. The PGKneo-loxP-3' HPRT cassette excised from pVNLH using SalI and AscI was cloned into the XhoI site and AscI site of VH21-12 (vector name: pMAC 3). Targeting vectors, target sequences and chromosomal alleles produced by homologous recombination are shown (FIG. 24).

[ A.2] transfection and isolation of G418-resistant clones

The culture of chicken DT40 cells was performed in RPMI1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco, hereinafter, FBS), 1% chicken serum (Gibco), and 10-4M 2-mercaptoethanol (Sigma). About 10 of DT40(MAC) -1⁷The cells were washed once in the non-supplemented RPMI1640 medium, suspended in 0.5ml of the non-supplemented RPMI1640 medium, added with 25. mu.g of the targeting vector pMAC3 linearized with the restriction enzyme NotI (TAKARA), transferred to a cuvette for electroporation (BioRad), and allowed to stand at room temperature for 10 minutes. The cuvette was set in a gene pulser (BioRad), and a voltage was applied under the conditions of 550V and 25. mu.F. After standing at room temperature for 10 minutes, the culture was carried out for 24 hours. The medium containing G418(1.5mg/ml) was replaced, and the mixture was injected into 2 96-well culture plates and subjected to selective culture for about 2 weeks. A total of 14 resistant colonies obtained by 2 transfections were separated and propagated for further analysis (clone name: DT40(MAC3))

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

In order to extract genomic DNA of the G418-resistant strain and select recombinants as templates, PCR was performed using the following primers to confirm whether recombination occurred site-specifically on mouse chromosome 11. The primer sequences are shown below.

m1117L (above)

Puro-1: 5'-GAGCTGCAAGAACTCTTCCTCACG-3' (Serial number 50)

kj neo (above)

m 116R (supra)

For PCR, Gene Amp9600 manufactured by Perkin-Elmer was used as a thermal cycler, LA Taq (TAKARA) was used as Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of 10 seconds at 98 ℃ and 9 minutes at 68 ℃ were carried out. As a result of PCR, 16 out of 17 clones were positive in all primer sets, and therefore, 2 clones were randomly selected from the 16 clones for subsequent analysis.

TABLE 23

[ A.3.2] Monochromatic FISH analysis

FISH analysis using mouse Cot-1DNA as a probe was carried out on 2 clones of DT40(MAC3) obtained as described above by Shinohara et al (Human Molecular Genetics, 10: 1163-.

Watch 24

[ A.3.3] two-color FISH analysis

Two-color FISH analysis was performed according to Songen et al (FISH protocol, Xiu run Co., 1994) from the above randomly selected DT40(MAC3) -160 and 187. FISH analysis using mouse cot-1DNA and mouse minor satellite DNA as probes revealed that mouse artificial chromosome MAC3 was present independently in 1 copy (fig. 25).

From these results, the following conclusions can be drawn: DT40 cell clone carrying mouse artificial chromosome vector MAC3 with loxP sequence inserted near mouse centromere as DNA insertion sequence was obtained.

[B] Introduction of MAC3 from Chicken DT40 cells containing mouse Artificial chromosome vector MAC3 into CHO cells

Mouse artificial chromosome vector MAC3 was introduced into CHO cells in order to stably insert loxP, which is the DNA sequence insertion site of mouse artificial chromosome vector MAC3, into a target gene or the like (for example, GFP gene or the like).

[ B.1] isolation of micronuclear cell fusion and drug-resistant clones

DT40(MAC3) -160, which is a recipient cell, was cultured in a cell culture dish, and when confluency reached, the cells were replaced with RPMI1640 medium supplemented with 20% FBS, 1% chicken serum, 10-4M 2-mercaptoethanol, and 0.05. mu.g/ml autumn formamide, and cultured for another 12 hours to form minicells. The culture medium was replaced with 24ml of serum-free DMEM medium, and 12 cells previously coated with 100. mu./ml polylysine were centrifuged at 25cm²2ml of each of the flasks (conical shape) was poured into the flask, and the flask was incubated at 37 ℃ for 30 minutes to attach the cells to the bottom of the flask. The supernatant was removed, and cytochalasin B (10. mu.g/ml, Sigma) solution previously incubated at 37 ℃ was filled into a flask for centrifugation, and centrifugation was performed at 8000rpm at 34 ℃ for 1 hour. Minicells were suspended in serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, the suspension was centrifuged at 1700rpm for 10 minutes and suspended in 5ml of serum-free DMEM medium.

As donor cells, CHOhprt-deficient cells (obtained from the Japan health science research resource Bank, accession number JCRB0218) namely CHO (HPRT) were used^-). The purified microkernel was resuspended in 2ml of a serum-free medium containing PHA-P (SIGMA), and gently inoculated into F12 medium (Invitrogen) from which the culture supernatant was removed [ 10% FBS was added ]]CHO cell of (a). The plates were incubated at 37 ℃ for 15 minutes. Removing supernatant, completely dissolving PEG1000(Wako) solution [ 5g of PEG1000 in serum-free DMEM medium, adding 1ml of dimethyl sulfoxide, and performing filtration sterilization]Fusion was correctly performed with 1ml for 1 min. Serum-free medium (DMEM) was washed 4 times with 4ml, and 5ml of normal CHO cell medium was added and cultured overnight. By usingPBS (-) cell surface washing 2 times, by trypsin treatment to disperse cells, seeding in 5 diameter 10cm cell culture dish, to make 800 u G/ml adding G418, for 3-4 weeks selection culture. A total of 12 resistant colonies obtained by 2 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: CHO (HPRT)^-；MAC3))。

[ B.2] screening of drug-resistant clones

[ B.2.1] PCR analysis

In order to extract genomic DNA of the G418-resistant strain as a template for selection of recombinants, PCR was performed using the following primers to confirm whether the mouse artificial chromosome vector MAC3 could be introduced into CHO cells. The primer sequences are shown below.

kj neo (above)

m 116R (supra)

m1117L (above)

Puro-1 (above)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 10 minutes were carried out. As a result of PCR, 6 out of 7 clones were positive in all the primer sets, and the 6 clones were used for subsequent analyses.

TABLE 25

[ B.2.2] Monochromatic FISH analysis

The CHO (HPRT) obtained above was subjected to the procedure described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC3) was subjected to FISH analysis using mouse Cot-1DNA as a probe, and it was confirmed that MAC3 was introduced into CHO cells at a ratio of 90% or more in 3 out of 6 clones (fig. 26).

Watch 26

From the above results, the following conclusions can be drawn: the mouse artificial chromosome vector MAC3 can be introduced into CHO cells.

[C] Introduction of MAC3 from CHO cell containing mouse Artificial chromosome vector MAC3 into mouse ES cell

In order to examine the stability of the mouse ES cells and the mouse artificial chromosome vector MAC3 in mouse individuals, a chimeric mouse and an offspring-transferred mouse containing the mouse artificial chromosome vector MAC3 were prepared by introducing the mouse artificial chromosome MAC3 into the mouse ES cells.

[ C.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) to be used as recipient cell^-(ii) a MAC3) -1 and 6 were cultured in a cell culture dish, and at the time of reaching confluence, the culture was changed to F12 medium supplemented with 20% FBS and 0.1. mu.g/ml autumn acrylamide, and after culturing for another 48 hours, the medium was changed to F12 medium supplemented with 20% FBS and 0.1. mu.g/ml autumn acrylamide, and then cultured overnight to form minicells. The culture solution was removed, and cytochalasin B (10. mu.g/ml, Sigma) solution previously incubated at 37 ℃ was filled in a flask for centrifugation, and centrifugation was performed at 8000rpm at 34 ℃ for 1 hour. Minicells were suspended in serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, the suspension was centrifuged at 2000rpm for 10 minutes and suspended in 5ml of serum-free DMEM medium. Minicells were suspended in 5ml serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, centrifugation was carried out at 2000rpm for 10 minutes.

Donor cells B6 (HPRT) which was a 6 TG-treated HPRT-deficient strain obtained from CLEA, Japan and used for ES cells of C57B 6-system mice was used^-) For the culture, 10% FCS, LIF (Muerin Leukemia inhibition Factor), 1 × 10% were added to DMEM (Dulbecco's Modified Eagle's Medium-high glucose: SIGMA)^-5M2-ME (2-mercaptoethanol: SIGMA), L-glutamine (3.5 g/ml: GIBCO), sodium pyruvate solution (3.5 g/ml: GIBCO), MEM non-essential amino acids (0.125 mM: GIBCO) in 5% CO₂The culture was carried out at 37 ℃. Mouse ES cells were washed 2 times with PBS (-), then the cells were dispersed by trypsin treatment, recovered in a culture medium supplemented with 10% FBS in DMEM medium, centrifuged at 1500rpm, the supernatant removed, resuspended in 5ml serum-free medium, gently added to serum-free medium containing the centrifuged particles of minicells, and centrifuged at 1200 rpm. Removing supernatant, dissolving PEG1000(Wako) solution [ 5g of PEG1000 completely dissolved in serum-free DMEM medium, adding 1ml of dimethyl sulfoxide, and filtering for sterilization]Fusion was correctly performed with 0.5ml for 1 min 30 sec. 13ml of serum-free medium (DMEM) was gently added thereto, and centrifugation was performed at 1200 rpm. The supernatant was removed, a normal culture solution of mouse ES cells was added, and G418-resistant mouse embryo fibroblasts treated with mitomycin were used as feeder cells, and seeded on 2 cell culture dishes of 10cm in diameter and cultured overnight. G418 was added to the cells so that the concentration of the cells became 250. mu.g/ml, and selection culture was performed for 3 to 4 weeks. A total of 28 resistant colonies obtained by 2 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: B6 (HPRT)^-；MAC3))。

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of the G418-resistant strain and select recombinants as templates, PCR was performed using the following primers to confirm whether site-specific cleavage occurred on mouse chromosome 11. The primer sequences are shown below.

kj neo (above)

m 116R (supra)

m1117L (above)

Puro-1 (above)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 10 minutes were carried out. As a result of PCR, 27 out of 28 clones were positive in all the primer sets, and the 27 clones were used for subsequent analyses.

Watch 27

[ C.2.2] Monochromatic FISH analysis

The B6 (HPRT) obtained above was subjected to the procedure described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC3), and as a result, it was confirmed that MAC3 was introduced into mouse ES cells at a ratio of 95% or more in 5 out of 16 clones.

From the above results, the following conclusions can be drawn: mouse artificial chromosome vector MAC3 can be introduced into mouse ES cells (fig. 27).

Watch 28

[D] Stability of mouse Artificial chromosome vector MAC3 in mouse ES cells

For the mouse ES clone obtained above (e.g., B6 (HPRT)^-(ii) a MAC3) -3, -s6, aboveA [ C ] is]Medium) was cultured in a non-selective culture medium of 0 to 100PDL for a long period of time, and then the proportion of cells carrying MAC1 obtained by FISH analysis was measured, and as a result, the carrying rate was 95% or more even in 100PDL (fig. 28).

From the above results, it was revealed that mouse artificial chromosome vector MAC3 was very stably maintained in mouse ES cells (in vitro) at a rate of 95% or more.

[E] Preparation of chimera mouse carrying mouse artificial chromosome vector MAC3

The ES cell clones obtained above were used to prepare chimera mice according to the method of (Gene targeting, Experimental medicine, 1995). As the host, morula obtained by male and female mating of mch (icr) (white, purchased from clean, japan) was used. The injected embryos are transplanted into a foster mother, and as a result, the born baby mice can be judged by gross color as being chimeras or the chimerism rate can be judged as the contribution rate of ES cells in the cells forming the individual relative to ICR embryonic cells.

60 embryos injected with B6 (HPRT; MAC3) clone (e.g., B6 (HPRT; MAC3) -ES (MAC3) -s6 obtained as described above) were transplanted into a foster mother, resulting in the production of 8 chimeric mice (dark brown portion of gross). Of the 8, 2 were male and 1 for 10% chimeric mice, 1 for 5% chimeric mice, 6 were female and 4 for 10% chimeric mice, 2 for 5% chimeric mice. That is, it was shown that the ES cell line (B6 HPRT-/-strain) carrying mouse artificial chromosome MAC3 carries a chimera-forming ability, i.e., the normal tissue of mouse individual carries an ability to undergo differentiation.

[F] As described in example 8, a chimeric mouse carrying the mouse artificial chromosome vector MAC3 and a wild-type mouse were mated to prepare a mouse system TC (MAC3) in which progeny transmitted MAC 3. In addition, the stability of MAC3 in somatic cells can be tested using the TC (MAC3) mouse system described above.

EXAMPLE 6 construction of mouse Artificial chromosome vector GFP-MAC

As an example of a gene encoding a useful protein in mouse artificial chromosome vector MAC3, EGFP, which is a fluorescent gene inserted using the Cre/loxP system, was examined for the expression and long-term stability of a functional protein (FIG. 5).

[A] Insertion of specific Gene (for example, GFP) contained in mouse Artificial chromosome vector MAC3 vector contained in CHO cell into mouse Artificial chromosome vector MAC3 by Cre/loxP System

It was examined whether loxP site works and plasmid DNA can be site-specifically inserted in mouse artificial chromosome vector MAC3 in which a PGKneo-loxP-3' HPRT type loxP site was inserted as a DNA insertion sequence in mouse artificial chromosome MAC.

[ A.1] preparation of EGFP insertion vector

The basic plasmid for inserting the loxP site was V913(Lexicon genetics). 5' HPRT-loxP oligo-synthesized loxP sequence was cloned into XbaI site of V820(Lexicon genetics). 5' HPRT-loxP was cloned into ClaI and AscI of V907(Lexicon genetics), and PGKhygro was cloned into ClaI and KpnI sites (vector name: pX6.1). HS4-CAG-EGFP-HS4 (donated by doctor Okagaku, Osaka university, and doctor Felsenfeld, NIH) excised from NotI and SalI was cloned into the NotI site and SalI site of X6.1, and used as a GFP insertion construct (vector name: pX6.1EGFP) of the HPRT reconstitution system. Chromosomal site-specific DNA generated by GFP insertion using the HPRT reconstitution system by Cre/loxP system insertion is shown in FIG. 29.

[ A.2] transfection and isolation of HAT-resistant clones

Gene transfer was carried out by lipofection, and Cre 1. mu.g and GFP insertion vector 2. mu.g were introduced into 6-well cells that reached 90% confluence according to a commercially available protocol (Invitrogen). When 2 weeks of culture were carried out under HAT selective culture, resistant colonies appeared, and a total of 22 colonies obtained by 2 introductions were separated and proliferated for subsequent analysis (clone name: CHO (GFP-MAC)).

[ A.3] screening of drug-resistant clones

[ A.3.1] confirmation of GFP inserts by fluorescence microscopy

As a result of observing 22 colonies of the clones under a fluorescence microscope, GFP-positive cells were observed in all the clones, and the positive rate was about 100%.

[ A.3.2] PCR analysis

In order to screen for recombinants using genomic DNA of the HAT-resistant strain as a template, PCR was performed using the following primers to confirm whether or not the insertion of the GFP gene occurred site-specifically. The primer sequences are shown below.

TRANS L1 (supra)

TRANS R1 (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer was used as a thermal cycler, Ampli Taq Gold (Applied Biosystems) was used as a Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after heat denaturation at 94 ℃ for 10 minutes, 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds were carried out. As a result of PCR, 22 clones were all positive, and the 22 clones were used for the subsequent analysis.

Watch 29

[ A.3.3] two-color FISH analysis

Of the 6 clones randomly selected from the above results, two-color FISH analysis was performed according to Songen et al (FISH protocol, Xiu run Co., 1994). FISH analysis was performed using mouse cot-1DNA and X6.1EGFP as probes, and it was confirmed that 3 out of 6 clones carried 1 copy of GFP-MAC at a rate of 50% or more, and that X6.1EGFP-derived signal was present, and that EGFP was site-specifically inserted because no signal was detected at MAC3 before site-specific insertion of EGFP as a negative control (FIG. 30).

Watch 30

By carrying the GFP gene on the mouse artificial chromosome MAC3 in the above experiment, GFP expression was observed, and it was confirmed that CHO cells carrying the mouse artificial chromosome vector GFP-MAC were obtained.

[B] Introduction of GFP-MAC into mouse ES cells from CHO cells containing mouse Artificial chromosome vector GFP-MAC

[ B.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (GFP-MAC) -4, 10, and 12 as recipient cells were cultured in a cell culture dish, and when confluency reached, the culture was changed to F12 medium supplemented with 20% FBS and 0.05. mu.g/ml autumn acrylamide, and after culturing for another 48 hours, the culture was changed to F12 medium supplemented with 20% FBS and 0.05. mu.g/ml autumn acrylamide, and then cultured overnight to form minicells. The culture solution was removed, and cytochalasin B (10. mu.g/ml, Sigma) solution previously incubated at 37 ℃ was filled in a flask for centrifugation, and centrifugation was performed at 8000rpm at 34 ℃ for 1 hour. Minicells were suspended in serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, the cells were cultured at 2000rpm for 10 minutes and suspended in 5ml of serum-free DMEM medium.

Minicells were suspended in 5ml serum-free DMEM medium and purified using 8 μm, 5 μm, 3 μm filters. After purification, centrifugation was carried out at 2000rpm for 10 minutes.

The donor cells used were wild type B6 cells and mouse ES cells of wild type TT2F cells, which were established from the ES cells of C57B6 system mice obtained from Clea, Japan. For the culture, 10% FCS, LIF (Mueri) was added to DMEM (Dulbecco's Modified Eagle's Medium-high glucose: SIGMA)n Leukemia InhibitoryFactor)、1×10^-5M2-ME (2-mercaptoethanol: SIGMA), L-glutamine (3.5 g/ml: GIBCO), sodium pyruvate solution (3.5 g/ml: GIBCO), MEM non-essential amino acids (0.125 mM: GIBCO) in 5% CO₂The culture was carried out at 37 ℃. Mouse ES cells were washed 2 times with PBS (-), then the cells were dispersed by trypsin treatment, recovered in a culture medium supplemented with 10% FBS in DMEM medium, centrifuged at 1500rpm, the supernatant was removed, resuspended in 5ml of serum-free medium, gently supplemented with serum-free medium containing the centrifuged particles of minicells, and centrifuged at 1200 rpm. Removing supernatant, dissolving PEG1000(Wako) solution [ 5g of PEG1000 completely dissolved in serum-free DMEM medium, adding 1ml of dimethyl sulfoxide, and filtering for sterilization]Fusion was correctly performed with 0.5ml for 1 min 30 sec. 13ml of serum-free medium (DMEM) was gently added thereto, and centrifugation was performed at 1200 rpm. The supernatant was removed, a normal culture solution of mouse ES cells was added, and G418-resistant mouse embryo fibroblasts treated with mitomycin were used as feeder cells, and seeded on 2 cell culture dishes of 10cm in diameter and cultured overnight. G418 was added to the cells so that the concentration of the cells became 250. mu.g/ml, and selection culture was performed for 3 to 4 weeks. A total of 36 resistant colonies obtained by 2 micronucleus cell fusions were proliferated and analyzed later (clone names: TT2F (GFP-MAC) and B6-ES (GFP-MAC)).

[ B.2] screening of drug-resistant clones

[ B.2.1] PCR analysis

In order to extract genomic DNA of the G418-resistant strain and select recombinants as templates, PCR was performed using the following primers to confirm whether site-specific cleavage occurred on mouse chromosome 11. The primer sequences are shown below.

TRANS L1: (above-mentioned)

TRANS R1: (above-mentioned)

m 116R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions of TRANS L1/R1 were such that after heat denaturation at 94 ℃ for 1 minute, 35 cycles of 98 ℃ for 10 seconds and 68 ℃ for 1 minute were carried out. The temperature and cycle conditions of TRANS L1/m 116R were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 7 minutes at 68 ℃ were carried out. As a result of PCR, 34 out of 36 clones were positive in all the primer sets, and 24 clones were randomly selected therefrom for the subsequent analysis.

Watch 31

[ B.2.2] Quinazine Hoechst double staining

Clones that were positive by the PCR analysis were doubly stained with quinacrine and Hoechst in the same manner as in the above method. When chromosome images of the clones after double staining with quinacrine Hoechst were observed with a fluorescence microscope, it was found that 18 of 24 clones carried mouse artificial chromosome GFP-MAC at a rate of 100%.

Watch 32

From the above results, the following conclusions can be drawn: mouse ES cells into which mouse artificial chromosome GFP-MAC has been introduced are of normal karyotype and can be used for long-term culture and production of chimeric mice.

[ B.2.3] two-color FISH analysis

The clones of mouse ES (GFP-MAC) obtained as described above were subjected to FISH analysis using mouse secondary satellite DNA and pX6.1E as probes by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001), and it was confirmed that GFP-MAC was introduced into mouse ES cells at a ratio of 95% or more in 6 out of 12 clones.

Watch 33

From the above results, the following conclusions can be drawn: the mouse artificial chromosome vector GFP-MAC can be introduced into mouse ES cells.

[C] Stability of mouse Artificial chromosome vector GFP-MAC in mouse ES cells

The proportion of GFP-MAC 3-carrying cells obtained by FISH analysis after long-term culture in non-selective culture at 0 to 100PDL was measured for the mouse ES clones obtained as described above (for example, B6-ES (MAC3) -9, obtained as described in [ B ] above), and the carrying rate was 95% or more even in 100PDL (FIG. 31, FIG. 32). Further, the colonies were observed under a fluorescent microscope, and as a result, GFP-positive cells were observed in all the clones, and the positive rate was about 100%.

Watch 34

From the above results, the following conclusions can be drawn: a foreign gene (such as EGFP gene) of 20kb or less can be site-specifically inserted into the mouse artificial chromosome vector MAC3 by using the Cre/loxP system, and MAC3 carrying the foreign gene is very stable in mouse ES cells, and the expression of the foreign gene on MAC3 is also stable for a long period of time.

[D] Preparation of chimeric mouse carrying mouse Artificial chromosome vector GFP-MAC

The ES cell clone obtained in [ B ] above was used to prepare a chimeric mouse according to the method of (Gene targeting, Experimental medicine, 1995). As the host, morula obtained by male and female mating of MCH (ICR) (white, purchased from Clea, Japan) and 8-cell stage embryo were used. The injected embryo is transplanted to a foster mother body, and the born young mouse judges whether the young mouse is a chimera or not according to the hair color.

Embryos (260 wild-type male B6(GFP-MAC) clones and 180 wild-type female TT2F (GFP-MAC) clones) into which wild-type male B6(GFP-MAC) clone and wild-type (GFP-MAC) TT2F female clones (for example, B6-ES (GFP-MAC)4 and 18, TT2F (GFP-MAC) -12, obtained as described above) were injected, respectively, were transplanted into the foster mother, and as a result, chimera mice were born (dark brown portions were confirmed in the hair color). 42 chimeric mice derived from male wild type B6(GFP-MAC) clones were born, 20 of which were male, 1 for GFP-positive 50%, 5 for 40%, 1 for 30%, 7 for 20%, 3 for 10% and 3 for 5%. In addition, 14 chimeric mice derived from wild type TT2F (GFP-MAC) clone were born, of which 1 was an individual with a mosaic rate of almost no white observed part of about 100% and GFP-positive.

As described above, it was shown that the ES cell lines (B6 and TT2F) carrying the mouse artificial chromosome vector GFP-MAC have chimera-forming ability, i.e., the normal tissues of the mouse individuals carry the ability to differentiate.

[E] Transfer of mouse artificial chromosome from progeny of chimeric mouse carrying mouse artificial chromosome vector GFP-MAC

Of 4 mice born as chimeras obtained by crossing female chimera mice (chimera rate of about 100%) prepared as described in [ D ] above with C57B6 (black, purchased from the company CLEA, Japan) male mice, 3 mice were dominant genetic characteristics of GFP-MAC derived from ES cells, and GFP fluorescence was observed. In addition, GFP fluorescence was observed in the whole body of 1 mouse out of 3 mice, indicating that the mouse artificial chromosome was stable even in the individual mouse (fig. 33). The mouse system that transmitted progeny with GFP-MAC was called TC (GFP-MAC). In addition, the stability of GFP-MAC in somatic cells can be examined using the TC (GFP-MAC) mouse system described above, as described in example 8.

EXAMPLE 7 stability of mouse Artificial chromosome vector MAC1

[ A.1] stability of mouse Artificial chromosome vector MAC1 in CHO cells

For the CHO clone obtained as described above (e.g., CHO (HPRT)^-(ii) a MAC1) -8, 22, obtained in example 2) were cultured for a long period in 25PDL in the presence of non-selective cells, and the proportion of cells carrying AC1 was measured by FISH analysis, and as a result, the carrying rate was 90% or more even in 25 PDL. On the other hand, CHO cells carrying GFP-carrying HAC vector (21HAC2) derived from chromosome 21 described in Kazuki et al (Gene Therapy: PMID: 21085194, 2010) have a carrying rate of 70% or less in 25 PDL. Representative results thereof are shown in fig. 34.

[ A.2] stability of mouse Artificial chromosome vector MAC1 in mouse ES cells

The mouse ES clones obtained as described above (for example, KO56(MAC1) -5 and TT2F (MAC1) -23 obtained as described in example 2) were carried at a carrying rate of 90% or more in 75PDL, as measured by the proportion of cells carrying MAC1 obtained by FISH analysis after a long-term culture in non-selective culture in 0 to 75 PDL. On the other hand, mouse ES cells carrying a GFP-carrying HAC vector (21HAC2) derived from chromosome 21 described in Kazuki et al (Gene Therapy: PMID: 21085194, 2010) had a carrying rate of 70% or less in 75 PDL. Representative results thereof are shown in fig. 35.

[ A.3] preparation of chimera mouse carrying mouse Artificial chromosome vector MAC1

Using the ES cell clone obtained in example 2 above, a chimera mouse was prepared by the method of Tomizuka et al (NatureGenet.16: 133, 1997). As the host, 8-cell stage embryos obtained by male-female mating of MCH (ICR) (white, purchased from Clea, Japan) were used. The injected embryos are transplanted to a foster mother, and as a result, the born mice judge whether the mice are chimeras or not through hair color. In the case of the ES clone carrying MAC1 (for example, KO56MAC1-5 and TT2FMAC1-4 obtained in example 2), 1620 embryos injected were transplanted into the foster mother, and 56 chimera mice (dark brown portions in the hair color) were born. Of these, 13 is only an individual in which the fitting rate of a portion where white color is hardly observed is about 100%. That is, it was shown that ES cell lines (KO56 and TT2F) carrying mouse artificial chromosome vector MAC1 carry chimera-forming ability, i.e., normal tissues of mouse individuals carry the ability to differentiate.

[ A.4] delivery of MAC1 from progeny of chimeric mice carrying the mouse artificial chromosome vector MAC1

2 female chimera mice (chimera ratio of about 100%) prepared as described in [ A.3] above were mated with MCH (ICR) (white, purchased from CLEA, Japan) male mice. Of the 18 mice born from chimeric mice, 13 showed dark brown color carrying a dominant genetic signature derived from ES cells. This indicates that the ES cell line carrying MAC1 was differentiated into functional egg cells in female chimera mice. In addition, the transfer of MAC1 was investigated by GFP fluorescence, and as a result, GFP was positive in 6 out of 13 (46%), and transfer of MAC1 in the progeny of chimeric mice was confirmed. That is, according to the mendelian genetic law, MAC1 was confirmed to appear at a frequency of about 50%, indicating that the carrying rate of MAC1 in ova was close to 100%. The mouse system that transmitted progeny with MAC1 is called TC (MAC 1).

[ A.5] stability of MAC1 in somatic cells of TC (MAC1) mouse System

[ A.5.1] Observation with a solid fluorescence microscope

In the TC (MAC1) mice obtained as described above, 1 of each of the brain, thymus, heart, lung, liver, kidney, spleen, small intestine, muscle, testis (or ovary) was observed under a solid fluorescence microscope, and GFP-positivity was observed in all tissues, and the positive rate was 100%. Representative results of the female (5) are shown in FIG. 36.

[ A.5.2] FACS analysis of blood System cells

The GFP-positivity rates in bone marrow and spleen cells were examined using B cells (CD19), T cells (CD4, CD8), and an antibody specific to megakaryocytes (CD41) (Becton, Dickinson and Company), and the positive rate was 95% or more in all tissues. On the other hand, mice carrying GFP-loaded HAC vector (21HAC2) derived from chromosome 21 described in Kazuki et al (Gene Therapy: PMID: 21085194, 2010) had a positive rate of 15% or less in all tissues. Representative results are shown in fig. 37 and 38.

[ A.5.3] Fluorescence In Situ Hybridization (FISH) analysis

Furthermore, FISH analysis using mouse secondary satellite DNA as a probe was carried out using tail fibroblasts prepared from the same individuals as described above by the method described in Shinohara et al (Human Molecular Genetics, 10: 1163-1175, 2001), whereby the presence of MAC1 was visually confirmed and the presence of MAC1 independent of mouse chromosome in 95% or more of the cells was confirmed (FIG. 39).

From the above results, it was confirmed that mouse artificial chromosome vector MAC1 was very stably maintained in a proportion of 90% or more in mouse ES cells (in vitro) and mouse tissues (in vivo).

EXAMPLE 8 preparation and stability of mouse carrying mouse Artificial chromosome vector CYP3A-MAC

[A] Transfer of CYP3A-MAC from CHO cells into mouse A9 cells

To prepare mouse ES cells carrying CYP3A-MAC, CYP 3A-MAC-carrying CHO cells (CHO (CYP3A-MAC, hCHr 7-. DELTA.CYP 3A)22, 26, 34, 35, etc.) obtained in example 3 above were introduced into mouse A9 cells having high minicell-forming ability with respect to mouse A9 cells by the micronocyte fusion method. A total of 25 resistant colonies obtained by 8 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: A9(CYP 3A-MAC)). As a result, 6 clones were positive in PCR using the primers described above, which detected only the CYP3A-MAC region. Furthermore, FISH analysis was performed using CYP3A-BAC (RP11757A13) (CHORI) and mouse minor satellite DNA probes (Tomizuka et al, NatureGenet.16: 133, 1997), and it was confirmed that 3 clones were among 6 clones in which the presence of CYP3A-MAC was specifically detected using the above probes (FIG. 40). From the above, the following conclusions can be drawn: a9 cells carrying CYP3A-MAC were obtained from 3 clones.

[B] Transfer of CYP3A-MAC from A9 cells into mouse ES cells

To prepare chimeric mice carrying CYP3A-MAC, the above-mentioned [ A ] was prepared by micronuclear cell fusion method]The obtained A9 cell carrying CYP3A-MAC was introduced into mouse ES cells (wild type TT 2F). The method according to Tomizuka et al (NatureGenet.16: 133, 1997) consists of a carrier carrying about 10⁸A9 cells (A9(CYP3A-MAC)8, 9, etc.) each containing CYP3A-MAC were purified and suspended in 5ml of DMEM. About 10 by trypsin treatment⁷Mouse ES cells TT2F were washed three times with DMEM, suspended in 5ml of DMEM, added to the centrifuged minicells, and centrifuged at 1250rpm for 10 minutes to completely remove the supernatant. The pellet was sufficiently disentangled by gently beating, and 1: 1.4PEG solution [ 5g of PEG1000(Wako pure chemical industries, Ltd.), 1ml of DMSO (Sigma) ] was added to 6ml of DMEM]0.5ml, and stirred well for about 1 minute and 30 seconds. Then, 10ml of DMEM was added slowly, the mixture was stirred at 1250rpm for 10 minutes, suspended in 30ml of ES medium, and poured into 3 petri dishes (cones) of 100mm diameter, which were previously seeded with feeder cells, to culture the cells. After 24 hours, the medium was replaced with a medium containing G418 at a concentration of 300. mu.g/ml, and selection culture was carried out for about 1 week. As a result, a total of 34 colonies were separated and proliferated for subsequent analysis. 14 clones from A9(CYP3A-MAC)8 and 7 clones from A9(CYP3A-MAC)9 were positive in PCR using the above primers which detected only the CYP3A-MAC region. Furthermore, FISH analysis was performed on 20 clones out of the above using DNA derived from CYP3A-BAC (RP11-757A13) (CHORI) (Tomizuka et al, Nature Genet.16: 133, 1997), and as a result, the above probes were specifically used for detection, and 8 clones with normal karyotype were obtained (FIG. 41). From the above, the following conclusions can be drawn: 8 grams can be obtainedCloned TT2F cells carrying CYP 3A-MAC.

[C] Stability in mouse ES cells of CYP3A-MAC

The mouse ES clones obtained in [ B ] (for example, TT2F (CYP3A-MAC)8-5, 8-22, 9-4, 9-7, and 9-9, obtained in [ B ]) were tested for the proportion of CYP 3A-MAC-carrying cells obtained by FISH analysis after long-term culture in non-selective culture in 0 to 100PDL, and as a result, the carrying rate was 95% or more even in 100 PDL. (FIG. 42).

[D] Preparation of chimeric mice carrying CYP3A-MAC

The CYP 3A-MAC-carrying ES cell clone obtained in [ B ] above was used to prepare a chimeric mouse by the method of Tomizuka et al (Nature Genet.16: 133, 1997). As the host, 8-cell stage embryos obtained by male-female mating of MCH (ICR) (white, purchased from Clea, Japan) were used. The injected embryo is transplanted to a foster mother, and the born baby mouse can judge whether the mouse is a chimera or not according to the hair color. 840 embryos injected with ES clones carrying MAC1 (for example, TT2F (CYP3A-MAC)8-5, 8-16, 8-22, 9-4, 9-7, 9-9, 9-10, etc. [ B ]) were transplanted into the foster mother, and 28 chimeric mice (dark brown portions were confirmed in the gross color) were born. Of these, 5 were individuals in which the chimeric ratio of the white-free portion was almost 100%. That is, it was shown that the ES cell line (TT2F) carrying the mouse artificial chromosome vector CYP3A-MAC carries a chimera-forming ability, i.e., normal tissues of individual mice carry an ability to undergo differentiation.

[E] CYP3A-MAC transmission from progeny of chimeric mice carrying CYP3A-MAC

5 female chimera mice (chimera ratio of about 100%) prepared according to [ D ] above were mated with MCH (ICR) (white, purchased from Clea, Japan). Of the 60 young mice born from the chimera mice, 50 showed dark brown color carrying the dominant genetic signature derived from the ES cells. This indicates that the ES cell line carrying CYP3A-MAC was differentiated into functional egg cells in female chimeric mice. In addition, the GFP fluorescence was used to study the CYP3A-MAC carrier, and as a result, the GFP-positive was observed in 29 out of 50 (58%), and the CYP3A-MAC carrier was confirmed in the progeny of the chimera mouse. That is, the occurrence of CYP3A-MAC characteristic was confirmed to be about 50% according to the Mendelian genetic code, indicating that the carrying rate of CYP3A-MAC in the ovum is close to 100%. The mouse system that passed progeny with CYP3A-MAC was called TC (CYP 3A-MAC).

[F] Stability of CYP3A-MAC in somatic cells of the TC (CYP3A-MAC) mouse System

[ F.1] Observation with solid fluorescence microscope

When 1 male (2) and 1 female (14) of the TC (CYP3A-MAC) mice obtained as described above were observed under a solid fluorescence microscope, GFP positivity was observed in all tissues, and the positive rate was 100%. Representative results of male (2) are shown in FIG. 43.

[ F.2] FACS analysis of blood System cells

The GFP positivity in bone marrow was examined using B cells (CD19), T cells (CD4, CD8), and an antibody specific to megakaryocytes (CD41) (Becton, Dickinson and Company), and the positivity was 94% or more in all tissues. On the other hand, mice carrying the HAC vector derived from chromosome 14 (CYP3A-HAC Δ) described in WO2009/063722(PCT/JP2008/068928) had a positive rate of 20% or less in all tissues. Representative results thereof are shown in fig. 44.

[ F.3] Fluorescence In Situ Hybridization (FISH) analysis

In addition, FISH analysis using CYP3A-BAC (RP11-757A13) DNA as a probe was carried out on individuals and tissues similar to those described above by the method described in Shinohara et al (Human molecular genetics, 10: 1163-1175, 2001), and the presence of CYP3A-MAC was visually confirmed and the presence of CYP3A-MAC was confirmed in 90-98% of the cells. On the other hand, in the case of the mice carrying the HAC vector derived from human chromosome 14 (CYP3A-HAC Δ) described in WO2009/063722(PCT/JP2008/068928), the positive rate was 56 to 97% in all tissues. Representative results thereof are shown in fig. 45.

[ F.4] transmissibility of TC (CYP3A-MAC) System

The transmission rate was investigated by mating 8 female TC (CYP3A-MAC) mice with 8 male MCH (ICR) (white, purchased from CLEA, Japan) mice. 81 mice were obtained, 38 were GFP negative and 43 were GFP positive (transmission rate 53%). That is, the transfer rate was in accordance with the Mendelian genetic code, and it was confirmed that the CYP3A-MAC characteristic appeared at a frequency of about 50%, indicating that the carrying rate of CYP3A-MAC in the ovum was close to 100%.

[ F.5] preparation and delivery rates of TC (CYP3A-MAC) homozygous System carrying 2 CYP3A-MAC

The TC (CYP3A-MAC) male mice and TC (CYP3A-MAC) female mice obtained above were mated, and an attempt was made to establish a TC (CYP3A-MAC) homozygous system carrying 2 CYP 3A-MACs. FISH analysis using CYP3A-BAC (RP11757A13) DNA as a probe was carried out using tail fibroblasts from 18 mice by the method described in Shinohara et al (Human Molecular Genetics, 10: 1163-1175, 2001), and the presence of CYP3A-MAC was visually confirmed. For the 4 × 36 system, 3 out of 12 mice had 2 copies, 5 had 1 copy, and 4 had 0 copies. For the 24 × 37 system, 1 was 2 copies, 3 was 1 copy, and 2 were 0 copies in 6 mice. A total of 18 CYP3A-MAC copies (4), 1 copy (8) and 0 copy (6) were obtained in a ratio of 1: 2: 1.5, essentially according to Mendelian's Law of inheritance. Representative results thereof are shown in fig. 46.

From the above results, it was confirmed that: CYP3A-MAC is very stable at a rate of 95% or more for a long period of time in mouse ES cells (in vitro), and is very stable at a rate of 90% or more in mouse tissues (in vivo), and is maintained in a homozygous system.

[G] Gene expression of tissue-specific CYP3A gene cluster in TC (CYP3A-MAC) mouse system

For 1 male (2) and female (14) of the TC (CYP3A-MAC) mice, total RNA was extracted from the brain, thymus, heart, lung, liver, kidney, spleen, small intestine, and muscle according to a commercially available protocol (QIAGEN), cDNA was synthesized according to a commercially available protocol (Invitrogen), and PCR was performed using the cDNA as a template to detect the expression of the human CYP3A gene cluster and the mouse CYP3a gene cluster. The primer sequences are shown below.

Primer for detecting human CYP3A gene cluster expression:

3A 4-1L: 5'-gtatggaaaagtgtggggct-3' (Serial number 51)

3A 4-1R: 5'-atacttcaagaattgggatg-3' (Serial number 52)

3A 4-2L: 5'-ccaagctatgctcttcaccg-3' (Serial number 53)

3A 4-2R: 5'-tgaagaagtcctcctaagct-3' (Serial number 54)

3A 5-1L: 5'-ctctgtttccaaaagatacc-3' (Serial number 55)

3A 5-1R: 5'-tcaacatctttcttgcaagt-3' (Serial number 56)

3A 7-1L: 5'-agcttttaagatttaatcca-3' (Serial number 57)

3A 7-1R: 5'-gagctttgtgggtctcagag-3' (Serial number 58)

3A 7-2L: 5'-ctctcagaattcaaaagact-3' (Serial number 59)

3A 7-2R: 5'-agaagaagtcctccaaagcg-3' (Serial number 60)

3A 43-2L: 5'-tatgacacaactagcaccac-3' (Serial number 61)

3A 43-2R: 5'-agtgtctagtgttctgggat-3' (Serial number 62)

Primers for detecting expression of mouse Cyp3a gene cluster:

3a 11-1L: 5'-tcaaacgcctctccttgctg-3' (Serial number 63)

3a 11-1R: 5'-gcttgcctttctttgccttc-3' (Serial number 64)

3a 11-2L: 5'-ggtaaagtacttgaggcaga-3' (Serial number 65)

3a 11-2R: 5'-agaaagggctttatgagaga-3' (Serial number 66)

3a 13-1L: 5'-agaaacatgaggcagggatt-3' (Serial number 67)

3a 13-1R: 5'-acaaggagacatttagtgca-3' (Serial number 68)

3a 13-2L: 5'-taccccagtatttgatgcac-3' (Serial number 69)

3a 13-2R: 5'-agataactgactgagccaca-3' (Serial number 70)

Control gene expression detection primers:

GAPDH-F: 5'-CCATCTTCCAGGAGCGAGA-3' (Serial number 71)

GAPDH-R: 5'-TGTCATACCAGGAAATGAGC-3' (Serial number 72)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, ExTaq (Takara Shuzo) was used as a thermal cycler, and a buffer and dNTPs (dATP, dCTP, dGTP, and dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 35 cycles were carried out with 1 cycle of 93 ℃ for 1 minute, 56 ℃ for 1 minute and 72 ℃ for 1 minute after 5 minutes of thermal denaturation at 93 ℃.

As a result, in the mice carrying TC (CYP3A-MAC), CYP3a4 was detected only in the liver and small intestine, CYP3a5 was detected only in the liver, small intestine and lung, CYP3a7 was detected only in the liver, small intestine, kidney and lung, CYP3a43 was detected only in the liver, small intestine and kidney, CYP3a11 was detected only in the liver and small intestine, and CYP3a13 was detected only in the liver and small intestine. In addition, control GAPDH was detected in all tissues. Representative results of the female (14) are shown in FIG. 47. Thus, tissue-specific expression as found in humans was confirmed, indicating that it was humanized.

[H] Gene expression of the time-specific CYP3A gene cluster in the TC (CYP3A-MAC) mouse system

Total RNA was extracted from livers of GFP-positive TC (CYP3A-MAC) mice at 14.5 days gestational age, 16.5 days gestational age, 18.5 days gestational age, 0 days postnatal, 4 weeks, 6 weeks, 12 weeks, and 24 weeks according to a commercially available protocol (QIAGEN), cDNA was synthesized according to a commercially available protocol (Invitrogen), PCR was performed using the cDNA as a template, and expression was detected using primers for detecting the expression of the human CYP3A gene cluster and the expression of the mouse CYP3a gene cluster.

As a result, it was confirmed that human CYP3a4, human CYP3a5, mouse CYP3a11 and mouse CYP3a13, which were expressed in an adult form, were strongly expressed in the mature stage, and CYP3a7, which was expressed in a fetal form, was strongly expressed in the fetus. In addition, GAPDH as a control was detected in the same level in all gestational age and week age. Representative results thereof are shown in fig. 48. Thus, it was confirmed that the expression was time-specific as found in human, and it was shown that the expression was humanized.

EXAMPLE 9 preparation of TC (CYP3A-MAC)/Δ CYP mouse System

[A] Construction of a mouse System carrying CYP3A-MAC and having both alleles of the endogenous Cyp3a Gene group disrupted

The TC (CYP3A-MAC) prepared in example 8 above was backcrossed with the Δ CYP system prepared in example 7 of WO2009/063722(PCT/JP2008/068928), and the genotype of the obtained GFP-positive mouse individual was analyzed by the PCR method described above. The tail of the mating young mouse 51 was partially cut off, and genomic DNA was prepared from this sample. The obtained DNA was subjected to PCR using the primer for detecting CYP3A-MAC and the primers described in table 1 of WO2009/063722(PCT/JP2008/068928) in the same manner as described above to detect the carryover of CYP3A-MAC and KO of the CYP3a gene cluster, and as a result, it was confirmed that CYP3A-MAC is carried in the 24 mouse system and one allele of the endogenous CYP3a gene group is disrupted (heterozygous (ヘテロ) KO). Further, mice carrying Cyp3A-MAC, in which the heterozygous Cyp3a gene group was disrupted, were backcrossed with the Δ Cyp system, and a part of the tail of 38 GFP-positive mice obtained was excised, and genomic DNA was prepared from the sample, and genotype analysis was performed by the same PCR method as described above. As a result, it was confirmed that CYP3A-MAC was carried in 18 mouse systems and both alleles of the endogenous CYP3a gene group were disrupted (heterozygous KO). (hereinafter, referred to as TC (CYP3A-MAC)/Δ CYP).

[B] Metabolic analysis in TC (CYP3A-MAC)/Δ CYP mouse System

The metabolites of alpha-OH-Triazolam and 4-OH-Triazolam were measured by mixing TC (CYP 3A-MAC)/delta CYP mice and liver microsomes of individual delta CYP mice with Triazolam (Triazolam) (200. mu.M) known to be metabolized by CYP3A4 according to Omura et al (J.biol.chem., 239, 2370, 1964). As described in WO2009/063722(PCT/JP2008/068928), it was confirmed that the activities of a mouse and a human (HLM: human liver microsome) of the same system were the same in TC (CYP 3A-MAC. DELTA.)/Δ CYP mice. As described above, it was confirmed that the human CYP3A gene on CYP3A-MAC is functional in TC (CYP3A-MAC)/Δ CYP mouse system and is equivalent to that of human.

[C] Therefore, liver microsomes derived from the TC (CYP3A-MAC)/Δ CYP mouse system are useful as a sample for examining the efficacy and toxicity of the first phase reaction in drug development. Since the TC (CYP3A-MAC)/Δ CYP mouse system can reproduce drug metabolism in humans, it can be used as a model mouse for in vivo experiments for investigating drug efficacy and toxicity of the first phase reaction in drug development.

EXAMPLE 10 preparation of rat carrying mouse Artificial chromosome vector CYP3A-MAC

[A] Engraftment of CYP3A-MAC from A9 cells into rat ES cells

To prepare chimeric rats carrying CYP3A-MAC, rESWIv3i-1, a rat ES cell transferred from the CYP 3A-MAC-carrying A9 cell obtained in example 8 to transferable progeny, was prepared by micronuclear cell fusion (Hirabayashi et al, Mol Reprod Dev.2010Feb; 77(2): 94.) is introduced. According to the method of Tomizuka et al (NatureGenet.16: 133, 1997), from about 10⁸A9 cells (A9(CYP3A-MAC)8, 9, etc.) carrying CYP3A-MAC were purified to give minicells, which were suspended in 5ml of DMEM. About 10 by trypsin treatment⁷Rat ES cells rESWIv3i-1 were washed 3 times with DMEM, suspended in 5ml of DMEM, added to the centrifuged minicells, and centrifuged at 1250rpm for 10 minutes to completely remove the supernatant. The pellet was sufficiently disentangled by gently beating, and 1: 1.4PEG solution [ 5g of PEG1000(Wako pure chemical industries, Ltd.), 1ml of DMSO (Sigma) ] was added to the pellet and dissolved in 6ml of DMEM]0.5ml, and stirred well for about 1 minute and 30 seconds. Then, 10ml of DMEM was gradually added, the mixture was centrifuged at 1250rpm for 10 minutes, the suspension was suspended in 30ml of ES medium, and 3 petri dishes (cones) with a diameter of 100mm, which were inoculated with feeder cells in advance, were each poured and cultured. After 24 hours, the medium was replaced with a medium containing G418 at a concentration of 300. mu.g/ml, and selection culture was carried out for about 1 week. As a result, a total of 10 colonies were separated and proliferated for subsequent analysis. 2 clones from A9(CYP3A-MAC)8, 3 clones from A9(CYP3A-MAC)8 were positive in PCR using the above primers detecting only the CYP3A-MAC region. Furthermore, FISH analysis was performed on the above 5 clones using CYP3A-BAC (RP11-757A13) (CHORI) and mouse Cot-1DNA (Tomizuka et al, Nature Genet.16: 133, 1997), and as a result, the above probes were specifically detected, and 3 clones with normal karyotype in rats were obtained (FIG. 49). From the above, the following conclusions can be drawn: rat ES cells carrying CYP3A-MAC were obtained from 3 clones.

[B] As described in example 8, rat ES cells carrying the mouse artificial chromosome vector CYP3A-MAC can be used to test stability in vitro. Furthermore, offspring-transferred rats were prepared from rat system rTC (CYP3A-MAC) which is a chimera rat prepared from the ES cells. In addition, the rTC (CYP3A-MAC) rat system described above can be used to test the stability of CYP3A-MAC in somatic cells. Liver microsomes derived from rTC (CYP3A-MAC) rat system are used as samples for examining the efficacy and toxicity of the first phase reaction in drug development. Since rTC (CYP3A-MAC) rat system can reproduce drug metabolism in humans, it can be used as a model rat for in vivo experiments for investigating drug efficacy and toxicity of the first-phase reaction in drug development.

EXAMPLE 11 construction of mouse Artificial chromosome vector hChr21q-MAC

In order to prepare a Down syndrome model mouse, hCHr21q-MAC was constructed in the same manner as in example 3 by transposing the mouse artificial chromosome vector MAC1 using the Cre/loxP system to clone a DNA sequence containing the region 33Mb more distal than the long-arm AP001657 of human chromosome 21.

[A] Introduction of hCHr21-loxP from DT40 containing hCHr21-loxP into CHO cells containing MAC1

A region more distal than AP001657 of the long arm of human chromosome 21 was inserted into mouse artificial chromosome vector MAC1 by transposition in CHO cells via a loxP site, and human chromosome 21, hCHR21-loxP, in which the loxP site was inserted into AP001657, was introduced into CHO cells containing mouse artificial chromosome vector MAC 1.

[ A.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) that is a CHOhprt deficient cell (obtained from the resource pool of health scientific research in Japan, accession number JCRB0218) containing MAC1 was treated in the same manner as described above using DT40 cells containing hCHhr 21-loxP and DT40(kk139) (Japanese unexamined patent publication No. 2007-295860) that are recipient cells^-(ii) a MAC1) was subjected to the micronucleus fusion method. A total of 114 resistant colonies obtained by 14 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: CHO (HPRT)^-；MAC1，hChr21-loxP))。

[ A.2] screening of drug-resistant clones

[ A.2.1] PCR analysis

In order to extract genomic DNA of hygromycin-resistant strains as templates for recombinant selection, PCR was performed on 60 out of the 114 clones using the following primers to confirm whether the human chromosome 21 fragment was introduced into CHO cells containing MAC 1. The primer sequences are shown below.

m115L (above)

EGFP (F) L (above)

kj neo (above)

m 116R (supra)

#21CEN <1> 2L: 5'-aaatgcatcaccattctcccagttaccc-3' (Serial number 73)

PGKr 1: 5'-ggagatgaggaagaggagaaca-3' (Serial number 74)

D21S 265-L: 5'-gggtaagaaggtgcttaatgctc-3' (Serial number 75)

D21S 265-R: 5'-tgaatatgggttctggatgtagtg-3' (Serial number 76)

D21S 261-L: 5'-gagggggactgggacaagccctttgctggaagaga-3' (Serial number 77)

D21S 261-R: 5'-acattaggaaaaatcaaaaggtccaattattaagg-3' (Serial number 78)

D21S 268-L: 5'-CAACAGAGTGAGACAGGCTC-3' (Serial number 79)

D21S 268-R: 5'-TTCCAGGAACCACTACACTG-3' (Serial number 80)

D21S 266-L: 5'-ggcttggggacattgagtcatcacaatgtagatgt-3' (Serial number 81)

D21S 266-R: 5'-gaagaaaggcaaatgaagacctgaacatgtaagtt-3' (Serial number 82)

D21S 1259-L: 5'-GGGACTGTAATAAATATTCTGTTGG-3' (Serial number 83)

D21S 1259-R: 5'-CACTGGCTCTCCTGACC-3' (Serial number 84)

CBR-L: 5'-gatcctcctgaatgcctg-3' (Serial number 85)

CBR-R: 5'-gtaaatgccctttggacc-3' (Serial number 86)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 11 clones out of 60 clones were positive in all the primer sets, and the 11 clones were used for subsequent analyses.

[ A.2.2] two-color FISH analysis

The CHO (HPRT) obtained above was subjected to the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC1, hChr21-loxP) was subjected to FISH analysis using mouse Cot-1DNA and human Cot-1DNA as probes, and it was confirmed that MAC1 and hChr21-loxP were introduced into CHO cells at a rate of 70% in 1 copy in 1 out of 1 of 6 clones (fig. 50).

From the above results, the following conclusions can be drawn: hCHr21-loxP can be introduced into CHO cells containing mouse artificial chromosome vector MAC 1.

[B]To CHO (HPRT)^-(ii) a MAC1, hChr21-loxP) cloning site-specific transposition of MAC1 vector at region 33Mb more distal than AP001657 of the long arm of human chromosome 21

To more stably maintain a region more distal than the 33Mb size DNA, i.e., AP001657 of the long arm of human chromosome 21, in a mouse individual, a transposition was inserted into mouse artificial chromosome vector MAC1 (fig. 51).

[ B.1] transfection and isolation of HAT-resistant clones

The CHO (HPRT) obtained above was subjected to lipofection^-(ii) a MAC1, hCHr21-loxP) -37 was introduced, and Cre 3. mu.g was introduced into 6-well cells that reached 90% confluence according to the commercially available protocol (Invitrogen). When 2 weeks of culture were carried out under HAT selection culture, resistant colonies appeared, and a total of 2 colonies obtained by 2 introductions were separated and proliferated for subsequent analysis (g)The Longming: CHO (hCHr21q-MAC, hCHr21-hCHr21 q)).

[ B.2] screening of drug-resistant clones

[ B.2.1] PCR analysis

In order to extract genomic DNA of the HAT-tolerant strain as a template and screen reciprocal transposition clones, PCR was performed using the following primers to confirm whether chromosomal reciprocal transposition was caused on the human chromosome 21 fragment and MAC 1. The primer sequences are shown below.

kj neo (above)

PGKr1 (supra)

D21S265-L (above)

D21S265-R (above)

D21S261-L (above)

D21S261-R (above)

D21S268-L (supra)

D21S268-R (supra)

D21S266-L (supra)

D21S266-R (above)

D21S1259-L (above)

D21S1259-R (above)

CBR-L (above)

CBR-R (above)

TRANS L1 (supra)

TRANS R1 (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 2 clones out of 2 clones were positive in all the primer sets, and the 2 clones were used for subsequent analyses.

[ B.2.2] two-color FISH analysis

FISH analysis using mouse Cot-1DNA and Human Cot-1DNA as probes was performed on 2 clones of CHO (hCHR21q-MAC, hCHR21-hCHR21q) obtained as described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-.

From the above results, the following conclusions can be drawn: the region 33Mb further from the long arm of human chromosome 21 than AP001657 can be cloned on mouse artificial chromosome vector MAC1 using reciprocal transposition.

[C] Transfer of hCHr21q-MAC from CHO cells into mouse ES cells

To prepare chimeric mice carrying hCHr21q-MAC, the chimeric mice were prepared from [ B ] above by micronuclear cell fusion]The obtained CHO cells carrying hCHr21q-MAC were introduced into mouse ES cells (wild type TT 2F). According to the method of Tomizuka et al (Nature Genet.16: 133, 1997), from about 10⁸CHO cells (CHO (hCHr21q-MAC, hCHr21-hCHr21q)1, 2) carrying hCHr21q-MAC were purified and suspended in 5ml of DMEM. About 10 by trypsin treatment⁷Mouse ES cells TT2F were washed 3 times with DMEM, suspended in DMEM5ml, added to the centrifuged minicells, and centrifuged at 1250rpm for 10 minutes to completely remove the supernatant. The pellet was sufficiently disentangled by gently beating, and 1: 1.4PEG solution [ 5g of PEG1000(Wako pure chemical industries, Ltd.), 1ml of DMSO (Sigma) was dissolved in 6ml of DMEM]0.5ml, and stirred well for about 1 minute and 30 seconds. Then, 10ml of DMEM was slowly added thereto, centrifuged at 1250rpm for 10 minutes, suspended in 30ml of ES medium, and poured into 3 petri dishes (cones) of 100mm in diameter, which were previously inoculated with feeder cells, to culture the cells. After 24 hours, the medium was replaced with a medium containing G418 at a concentration of 300. mu.g/ml, and selection culture was carried out for about 1 week. As a result, a total of 24 colonies were isolated andthe cells were proliferated and analyzed later. 2 clones from CHO (hCHr21q-MAC, hCHr21-hCHr21q)1, 6 clones from CHO (hCHr21q-MAC, hCHr21-hCHr21q)2 were positive in PCR using the above primers which detected only the hCHr21q-MAC region. Furthermore, FISH analysis was performed on 8 clones among the above using human Cot-1DNA and mouse minor satellite DNA (Tomizuka et al, Nature Genet.16: 133, 1997), and as a result, they were specifically detected using the above-mentioned probe and 2 clones with normal karyotype were obtained (FIG. 53). The following conclusions can be drawn as above: TT2F cells carrying hCHr21q-MAC were obtained from 2 clones.

[D] Stability in mouse ES cells of hCHr21q-MAC

The mouse ES clone obtained as described above (for example, TT2F (hCHr21q-MAC)22, obtained in [ C ]), was cultured for a long period in a non-selective culture of 0 to 50PDL, and then the proportion of cells carrying hCHr21q-MAC was measured by FISH analysis, and as a result, the carrying rate was 95% or more even in 50 PDL. (FIG. 54).

[E] Preparation of chimeric mouse carrying hCHr21q-MAC

Using the hCHr21 q-MAC-carrying ES cell clone obtained in the above [ C ], a chimera mouse was prepared by the method of Tomizuka et al (Nature Genet.16: 133, 1997). As the host, 8-cell stage embryos obtained by male-female mating of MCH (ICR) (white, purchased from Clea, Japan) were used. The injected embryo is transplanted to a foster mother, and the born baby mouse can judge whether the mouse is a chimera or not according to the hair color. 220 embryos injected with ES clones carrying MAC1 (for example, TT2F (hCHr21q-MAC)20, 22, etc., obtained in [ C ] above) were transplanted into the foster mother, and 18 chimera mice (dark brown portions in the hair color) were born. Of these, 2 was only an individual in which the chimerism rate of the white-barely observed portion was about 100%. Of these, 1 was a GFP-positive individual (FIG. 55). That is, it was shown that the ES cell line (TT2F) carrying the mouse artificial chromosome vector hCHr21q-MAC carries a chimera-forming ability, i.e., the normal tissue of the mouse individual carries an ability to undergo differentiation.

[F] As described in example 8, a mouse system TC (hCHR21q-MAC) in which hCHR21q-MAC was transferred as a progeny was prepared from chimeric mice carrying the mouse artificial chromosome vector hCHR21 q-MAC. In addition, the stability of hCHr21q-MAC in somatic cells can be tested using the TC (hCHr21q-MAC) mouse system described above. In addition, the TC (hCHr21q-MAC) system can be used as a model mouse of Down syndrome, and can be used for mechanism elucidation of symptomatic complications of Down syndrome and development of therapeutic drugs for improving symptoms.

EXAMPLE 12 construction of mouse Artificial chromosome vector hChr21q22.12-MAC

In order to prepare mice in which Down syndrome also appeared, hCH21q22.12-MAC was constructed in the same manner as in example 3 by transposing the mouse artificial chromosome vector MAC1 using the Cre/loxP system to clone a DNA sequence containing a region more distal than AP00172 in the long arm of human chromosome 21.

[A] Site-specific insertion of loxP site into AP001721 of human chromosome 21

In order to be inserted into the mouse artificial chromosome vector MAC1 by transposition through a loxP sequence, a loxP sequence was inserted into AP001721 proximal to DSCR (down syndrome-causing region) of human chromosome 21 (hChr21) in DT40 cells.

[ A.1] preparation of targeting vector pCKloxyphyg

A targeting vector pCKloxyphyg for inserting a recognition sequence loxP of Cre recombinase into the Down syndrome pathogenic gene region (DSCR) located near AP001721 on human chromosome 21 and on the centromere side (about 50Kb centromere side) was prepared as follows. First, the AP001721 genomic region was amplified by PCR using the following primers.

AML 5'. L1; 5'-TAGAATTCGTAGGCTTGGAAGCAGTGAGAGAGAA-3' (Serial number 87)

AML 5'. R2; 5'-GAAGACTGGTAAATCTGGTGGCTGTC-3' (Serial number 88)

AML 5'. L4; 5'-ATTAGATCTCCTGCTGTTATCTCATGCACTCTCA-3' (Serial number 89)

AML 5'. R4; 5'-ATTAGATCTATGATGCCTGATACATGGTCTGTGA-3' (Serial number 90)

The basic plasmid for inserting the loxP site was V901(Lexicon genetics). For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (Takara Shuzo) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 5 minutes. After subjecting the PCR product to protease K (Gibco) treatment, the PCR fragments (2.9kb and 2.0kb) were subjected to gel filtration using CHROMASPIN-TE400 (Clontech). Then, it was cleaved with restriction enzymes EcoRI (ニツポンジ - ン) and BglII (ニツポンジ - ン) and subjected to gel filtration using CHROMASPIN-TE1000 (Clontech). Next, the MC1-TK sequence was excised from V830(Lexicon genetics) by using Rsr II (NEB), and cloned into the recognition site of the restriction enzyme HindIII of the V901 plasmid (V901T-1). The PCR fragments (2.9kb and 2.0kb) were cloned into the EcoRI and BglII sites of the V901T-1 plasmid (V901T-1HR 2). Next, 5 '-HPRT-loxP-Hyg was excised from the 5' -HPRT-loxP-Hyg-TK vector described in Kazuki et al, a report (Gene Therapy: PMID: 21085194, 2010) using KpnI and AscI, and cloned into AscI and KpnI sites (pCKloxyphyg) of V901T-1HR 2. The size of the final loxP insert construct is 11.2 kb. Targeting vectors, target sequences and chromosomal alleles produced by homologous recombination are shown (FIG. 56).

[ A.2] transfection and isolation of hygromycin-resistant clones

The targeting vector pCKloxypryg prepared as described above was linearized with restriction enzymes NotI (TAKARA) and then transfected into DT40 hybrid cells carrying human chromosome 21 (BBRC 2004, Kazuki et al, DT40(21-2-3)), which were replaced with a medium containing hygromycin B (1.5mg/ml), and the cells were individually plated into 3 96-well plates and selectively cultured for about 2 weeks. 178 resistant colonies in total obtained by 4 transfections were separated and propagated for subsequent analysis (clone name: DT40 (hChr21q22.12-loxP)).

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

Genomic DNA was extracted from the hygromycin-resistant clones using the Puregene DNA Isolation Kit (Gentra System Co.), and homologous recombinants were identified by PCR using the following 2 sets of primers.

Identification of homologous recombinants was performed by PCR using the following 2 sets of primers.

AMLloxP-4L; 5'-AGAAAGGCAGGTGAGTGTGGAGGTAGA-3' (Serial number 91)

AMLloxP-4R; 5'-GAAGTGGGCTCACAGGAATTTTCCAA-3' (Serial number 92)

AMLloxP-8L; 5'-GGGCCTCTTTATTTGGCAGAATATCACC-3' (Serial number 93)

AMLloxP-8R; 5'-TTACACTGAGATTCAGGGCACGATGA-3' (Serial number 94)

For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 4 minutes at 68 ℃ were carried out. 178 clones were screened and 71 clones were identified as homologous recombinants.

[ A.3.2] southern blot analysis

Southern blot analysis was performed on 10 clones whose recombination was confirmed by the above PCR analysis as follows. The genomic DNA was treated with restriction enzyme EcoRI (TAKARA), electrophoresed in 0.8% agarose gel, and subjected to alkali blotting onto GeneScreen plus (TM) hybridization transfer membrane (NENTM Life Science Products, Inc.). For this filter, DNA hybridization was performed using an SP7 probe in which the gene sequence in AP001721 was amplified by PCR, and identification of homologous recombinants was performed. Preparation of SP7 Probe the following primers were used and DT was used40(21-2-3) as a template, and the PCR product was prepared by the random primer method using the PCR product as a template³²p-labeled DNA probes (Amersham, according to the protocol noted).

Primer for producing SP7 probe:

SP 7L; 5'-CAGCTGGGAAACACTGAGCAAGATTATG-3' (Serial number 95)

SP 7R; 5'-CTGCTAGACTGAAAATGCGTTTCCTCTG-3' (Serial number 96)

For PCR, Gene Amp9600 manufactured by Perkin-Elmer, EXTTaq (TAKARA) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after the heat denaturation at 93 ℃ for 5 minutes, 35 cycles of 1 cycle of 93 ℃ for 1 minute, 54 ℃ for 1 minute and 72 ℃ for 1 minute were carried out. It was predicted that a band of about 7.5kb was detected in non-homologous recombinants and about 9.4kb was detected in homologous recombinants by DNA hybridization (FIG. 56). As a result of DNA hybridization, all of the 10 clones were targeted homologous recombinants. Representative results thereof are shown in fig. 57.

[ A.3.3] two-color FISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of 10 clones among the above clones confirmed to be recombined using human cot-1DNA and hygromycin as probes, human chromosome 21 was not transposed to the host chromosome in all clones, and a hygromycin-derived signal was detected in the vicinity of 21q22, thereby confirming that recombination was site-specifically caused (FIG. 58). From the above results, the following conclusions can be drawn: the loxP site, which is a gene introduction site, is specifically inserted into the human chromosome 21 fragment.

[B] Introduction of hChr21q22.12-loxP into CHO cells containing MAC1 from DT40 containing hChr21q22.12-loxP

In order to transpose the region more distal than the long arm of human chromosome 21 AP001721 into mouse artificial chromosome vector MAC1 via the loxP site in CHO cells, hChr21q22.12-loxP, which is human chromosome 21 and is obtained by inserting the loxP site into AP001721, was introduced into CHO cells containing mouse artificial chromosome vector MAC 1.

[ B.1] isolation of micronuclear cell fusion and drug-resistant clones

The CHOhprt deficient cells (obtained from the resource pool of health scientific research in Japan, accession number JCRB0218) containing MAC1, namely CHO (HPRT), were treated in the same manner as described above using DT40(hChr21q22.12-loxP)47, a DT40 cell containing hChr21q22.12-loxP, which is a recipient cell^-(ii) a MAC1) was subjected to the micronucleus fusion method. A total of 140 resistant colonies obtained by 15 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: CHO (HPRT)^-；MAC1，hChr21q22.12-loxP))。

[ B.2] screening of drug-resistant clones

[ B.2.1] PCR analysis

In order to extract genomic DNA of hygromycin-resistant strains as templates for recombinant selection, PCR was performed on 20 out of the 140 clones using the following primers to confirm whether human chromosome 21 fragment was introduced into CHO cells containing MAC 1. The primer sequences are shown below.

m 115L (above)

EGFP (F) L (above)

kj neo (above)

m 116R (supra)

D21S 265-L: gggtaagaaggtgcttaatgctc (Serial number 97)

D21S 265-R: tgaatatgggttctggatgtagtg (Serial number 98)

D21S 261-L: gagggggactgggacaagccctttgctggaagaga (Serial number 99)

D21S 261-R: acattaggaaaaatcaaaaggtccaattattaagg (Serial number 100)

D21S 268-L: CAACAGAGTGAGACAGGCTC (Serial number 101)

D21S 268-R: TTCCAGGAACCACTACACTG (Serial number 102)

D21S 266-L: ggcttggggacattgagtcatcacaatgtagatgt (Serial number 103)

D21S 266-R: gaagaaaggcaaatgaagacctgaacatgtaagtt (Serial number 104)

D21S 1259-L: GGGACTGTAATAAATATTCTGTTGG (Serial number 105)

D21S 1259-R: CACTGGCTCTCCTGACC (Serial number 106)

CBR-L: gatcctcctgaatgcctg (Serial number 107)

CBR-R: gtaaatgccctttggacc (Serial number 108)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 13 out of 20 clones were positive in all the primer sets, and the 13 clones were used for subsequent analyses.

[ B.2.2] two-color FISH analysis

The CHO (HPRT) obtained above was subjected to the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC1, hchr21q22.12-loxP) was subjected to FISH analysis using mouse Cot-1DNA and human Cot-1DNA as probes, and it was confirmed that 2 clones out of the 6 clones introduced MAC1 and hchr21q22.12-loxP into CHO cells at a rate of 75% in 1 copy (fig. 59).

From the above results, the following conclusions can be drawn: hChr21q22.12-loxP can be introduced into CHO cells containing mouse artificial chromosome vector MAC 1.

[C]To CHO (HPRT)^-(ii) a MAC1, hchr21q22.12-loxP) clone site-specific transposition of MAC1 vector at region 12Mb more distal than AP001721 of long arm of human chromosome 21

To stably maintain a region more distal than AP001721 of the long arm of human chromosome 21, which is 12 Mb-sized DNA, in a mouse, a transposition was inserted into mouse artificial chromosome vector MAC1 (FIG. 60)

[ C.1] transfection and isolation of HAT-resistant clones

The CHO (HPRT) obtained above was subjected to lipofection^-(ii) a MAC1, hChr21q22.12-loxP) -12, 13 was genetically introduced, and Cre 3. mu.g was introduced into 6-well cells that reached 90% confluence according to the commercially available protocol (Invitrogen). When cultured for 2 weeks under HAT selective culture, resistant colonies appeared, and a total of 19 colonies obtained by 2 introductions were separated and propagated for subsequent analysis (clone name: CHO (hCHR21q22.12-MAC, hCHR 21-hCHR21q22.12)).

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of the HAT-tolerant strain as a template and screen reciprocal transposition clones, PCR was performed using the following primers to confirm whether chromosomal reciprocal transposition was caused on the human chromosome 21 fragment and MAC 1. The primer sequences are shown below.

kj neo (above)

PGKr1 (supra)

D21S265-L (above)

D21S265-R (above)

D21S261-L (above)

D21S261-R (above)

D21S268-L (supra)

D21S268-R (supra)

D21S266-L (supra)

D21S266-R (above)

D21S1259-L (above)

D21S1259-R (above)

CBR-L (above)

CBR-R (above)

TRANS L1 (supra)

TRANS R1 (supra)

For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 8 out of 19 clones were positive in all the primer sets, and the 8 clones were used for subsequent analyses.

[ C.2.2] two-color FISH analysis

FISH analysis was performed on the 8 clones of CHO (hCH21q22.12-MAC, hCHR 21-hCH21q22.12) obtained as described above by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001) using mouse Cot-1DNA and Human Cot-1DNA as probes, and it was confirmed that signals derived from Human chromosome 21 were observed at a ratio of 85% or more in MAC1 among 8 clones out of 8 clones (FIG. 61).

From the above results, the following conclusions can be drawn: the 12Mb region further from the long arm AP001721 of human chromosome 21 allows the cloning of the mouse artificial chromosome vector MAC1 using reciprocal transposition.

[D] Transfer of hChr21q22.12-MAC from CHO cells into mouse ES cells

To prepare chimeric mice carrying hChr21q22.12-MAC, the above-mentioned [ C ] was fused by micronuclear cell fusion method]Obtained carryingCHO cells with hChr21q22.12-MAC were introduced into mouse ES cells (wild type TT 2F). According to the method of Tomizuka et al (Nature Genet.16: 133, 1997), from about 10⁸Each of CHO cells (CHO (hCH21q22.12-MAC, hCHR 21-hCH21q22.12) 1, 12, etc.) carrying hCHR21q-MAC was purified and suspended in 5ml of DMEM. About 10 by trypsin treatment⁷Mouse ES cells TT2F were washed 3 times with DMEM, suspended in 5ml of DMEM, added to the centrifuged minicells, and centrifuged at 1250rpm for 10 minutes to completely remove the supernatant. The pellet was sufficiently disentangled by gently beating, and 1: 1.4PEG solution [ 5g of PEG1000 (Wako pure chemical industries, Ltd.), 1ml of DMSO (Sigma) was dissolved in 6ml of DMEM]0.5ml, and stirred well for about 1 minute and 30 seconds. Then, 10ml of DMEM was added slowly, the mixture was centrifuged at 1250rpm for 10 minutes, suspended in 30ml of ES medium, and then poured into 3 petri dishes (cones) of 100mm diameter, which were previously inoculated with feeder cells, for culture. After 24 hours, the medium was replaced with a medium containing G418 at a concentration of 300. mu.g/ml, and selection culture was carried out for about 1 week. As a result, a total of 13 colonies were separated and proliferated for subsequent analysis. 1 clone from CHO (hCH21q22.12-MAC, hCHR 21-hCH21q22.12) 1, 1 clone from CHO (hCH21q22.12-MAC, hCHR 21-hCH21q22.12) 12 were positive in PCR using the above primers detecting only the hCH21q22.12-MAC region. Furthermore, FISH analysis was performed on 2 clones among the above using human Cot-1DNA and mouse minor satellite DNA (Tomizuka et al, NatureGenet.16: 133, 1997), and as a result, the above probes were specifically detected and 1 clone with normal karyotype of the mouse was obtained (FIG. 62). The following conclusions can be drawn as above: TT2F cells carrying hChr21q22.12-MAC were obtained from 1 clone.

[E] Stability in mouse ES cells of hChr21q22.12-MAC

The mouse ES clone (for example, TT2F (hCh21q22.12-MAC) 8 obtained in [ D ] above) obtained in the above-mentioned manner was observed to have a carrying rate of 95% or more in 50PDL, as measured by the proportion of hCh21q22.12-MAC-carrying cells obtained by FISH analysis after a long-term culture in non-selective culture in 0 to 50 PDL. (FIG. 63).

[F] Production of chimeric mice carrying hChr21q22.12-MAC

The hChr21q22.12-MAC-carrying ES cell clone obtained in the above-mentioned [ D ] was used to prepare a chimera mouse by the method of Tomizuka et al (Nature Genet.16: 133, 1997). As the host, 8-cell stage embryos obtained by male-female mating of MCH (ICR) (white, purchased from Clea, Japan) were used. The injected embryo is transplanted to a foster mother, and the born baby mouse can judge whether the mouse is a chimera or not according to the hair color. 80 embryos injected with ES clones carrying hChr21q22.12-MAC (for example, TT2F (hChr21q22.12-MAC)8, obtained in [ D ] above) were transplanted into the foster mother, and as a result, 43 chimeric mice (dark brown portion in gross color) were born. Of these, 3 was only an individual in which the degree of engagement of the white-barely observed portion was about 100%. That is, it was shown that the ES cell line (TT2F) carrying the mouse artificial chromosome vector hChr21q22.12-MAC carries the chimera-forming ability, i.e., the normal tissue of the mouse individual carries the ability to differentiate.

[G] As described in example 8, a mouse system TC (hCH21q22.12-MAC) whose progeny transferred hCH21q22.12-MAC was prepared from chimeric mice carrying the mouse artificial chromosome vector hCH21q22.12-MAC. In addition, the stability of hCHR21q22.12-MAC in somatic cells can be examined using the TC (hCHR21q22.12-MAC) mouse system described above. In addition, the hChr21q22.12-MAC system can be used as a model mouse of Down syndrome, and can be used for mechanism elucidation of symptomatic complications of Down syndrome and development of therapeutic drugs for improving symptoms. In addition, the causative gene region of Down syndrome can be identified by comparing the phenotypes of the TC (hCHR21q-MAC) mouse system and the TC (hCHR21q22.12-MAC) mouse system.

EXAMPLE 13 stability of mouse Artificial chromosome vector MAC2

[A] Stability in CHO cells of mouse Artificial chromosome vector MAC2

For the CHO clone obtained as described above (e.g., CHO (HPRT)^-(ii) a MAC2) -13, 18, obtained in example 4 above), for non-PDL of 0 to 25The proportion of MAC 2-carrying cells obtained by FISH analysis after long-term culture in selective culture was measured, and the result showed a carrying rate of 90% or more even in 25 PDL. On the other hand, CHO cells carrying GFP-carrying HAC vector (21HAC2) derived from chromosome 21 described in Kazuki et al, report (Gene therapy: PMID: 21085194, 2010) had a carrying rate of 70% or less in 25 PDL. Representative results thereof are shown in fig. 64.

EXAMPLE 14 construction of mouse Artificial chromosome vector FVIII-MAC

As an example of a gene encoding a useful protein in mouse artificial chromosome vector MAC2, Factor VIII (FVIII) which is a pathogenic gene for hemophilia A can be inserted using Cre/loxP system and tested for expression and long-term stability of functional proteins.

[A] Insertion of a Gene encoding a specific useful protein (e.g., FVIII) into mouse Artificial chromosome vector MAC2 in CHO cells containing mouse Artificial chromosome vector MAC2 vector Using Cre/loxP System

It was examined whether loxP site works and circular DNA can be site-specifically inserted in mouse artificial chromosome vector MAC2 in which 5' HPRT-loxP-PGKhyg type loxP site was inserted as a DNA insertion sequence in mouse artificial chromosome MAC.

[ A.1] preparation of FVIII insertion vector

The promoter and polyA regions of pCAGGS (donated by doctor okada, university of Osaka) were cut with SalI and PstI sites, and cloned into SalI and PstI sites (pB-CAG) of pB3in which the multicloning site of pBluescript KS (-) (Stratagene) was changed. The B domain-deficient FVIII cDNA in pKF17K plasmid (presented by professor sakaguchi, university of autonomous medical science) was cut at XhoI and SalI sites and cloned into EcoRI site (pB-CAGF8) between promoter and polyA in pB-CAG. The CAG-F8-pA region of pB-CAGF8 was isolated via the SalI and AvrII sites and cloned into the SalI and AvrII sites (pB3-F8ins2) in a manner sandwiched between 2 HS4 insulator sequences on pB3ins 2. Subsequently, pPAC4 (Children's Hospital Oakland Research Institute (CHORI), BAC/PAC Resources) was introduced into the vector into which the 3' HPRT-loxP sequence was inserted. The AscI and FseI regions of pB3-F8ins2, the FVIII expression cassette (HS4-CAG-F8-pA-HS4), were cloned into the AscI and FseI sites of pPAC4 as a1 copy FVIII insert construct (vector name: pPAC4F8ins2H3-9(1 copy FVIII-PAC)) for HPRT reconstitution system (FIG. 65). Using the characteristics of compatible cohesive ends of the AvrII site and the NheI site, a 2-copy FVIII-PAC with 2 expression cassettes was obtained by recloning the AscI in a 1-copy FVIII-PAC from the AscI in the same vector to the NheI site from the 1 expression cassette region of AvrII 1. In addition, PAC vectors with up to 2, 4, 8, 16 copies of FVIII expression cassettes were also made by recloning the insert cassettes at the AscI and AvrII, vector side at the AscI and NheI sites (fig. 66).

[ A.2] transfection and isolation of HAT-resistant clones

Gene transfer was carried out by lipofection, and for 6-well cells that reached 90% confluence, Cre 1. mu.g and 1-copy FVIII-PAC vector 10. mu.g were introduced into CHO (HPRT) described above according to the protocol commercially available (Invitrogen)^-(ii) a MAC2) -13 and Kazuki et al (Gene Therapy: PMID: 21085194, 2010) as described in CHO (HPRT)^-(ii) a 21HAC2) (clone name: CHO (FVIIIx1-MAC) and CHO (FVIIIx 1-HAC)). In addition, Cre 1. mu.g and 16 copies of FVIII-PAC vector 10. mu.g were added to the above CHO (HPRT) according to the protocol of the commercial product (Invitrogen)^-(ii) a MAC2) -13 introduction (clone name: CHO (FVIIIx 16-MAC)). When 2 weeks of culture were carried out under HAT selective culture, resistant colonies appeared, and a total of 83 colonies were isolated and propagated for each of 46 clones obtained by 4 introductions for CHO (FVIIIx1-MAC), 18 clones for CHO (FVIIIx16-MAC), and 19 clones for CHO (FVIIIx1-HAC), and the subsequent analyses were carried out.

[ A.3] screening of drug-resistant clones

[ A.3.1] PCR analysis

In order to screen for recombinants using genomic DNA of HAT-resistant strains as templates, PCR was performed using the following primers to confirm whether or not the FVIII gene was site-specifically inserted. The primer sequences are shown below.

TRANS L1 (supra)

TRANS R1 (supra)

FVIIIF: 5'-ATACAACGCTTTCTCCCCAA-3' (Serial number 109)

FVIIIR: 5'-TCTTGAACTGAGGGACACTG-3' (Serial number 110)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, ExTaq (applied biosystems) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after heat denaturation at 94 ℃ for 10 minutes, 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds and 72 ℃ for 1 minute were carried out. As a result of PCR, the 24 clones were used for the subsequent analyses, which were positive for 4 clones derived from CHO (FVIIIx1-MAC), positive for 17 clones derived from CHO (FVIIIx1-HAC) and positive for 3 clones derived from CHO (FVIIIx 16-MAC).

[ A.3.2] Monochromatic FISH analysis

From the above results, among 7 clones, monochromatic FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). FISH analysis was performed using mouse cot-1DNA or human cot-1DNA as a probe, and as a result, 1 clone carried FVIII-MAC at a rate of 90% or more for CHO (FVIIIx1-MAC) source, 2 clones carried FVIII-HAC at a rate of 90% or more for CHO (FVIIIx1-HAC) source, and 1 clone carried FVIII-HAC at a rate of 90% or more for CHO (FVIIIx16-MAC) source.

[B] Gene expression analysis of FVIII Gene in CHO cells

In order to examine whether FVIIImRNA is expressed, RNA was extracted and cDNA synthesis was performed using the above described CHO (FVIIIx1-MAC)1-3 carrying FVIIIx1-MAC, CHO (FVIIIx1-HAC)1-2 carrying FVIIIx1-HAC, and CHO (FVIIIx16-MAC)16-1, 16-2, and 16-3 carrying FVIIIx16-MAC, and the following primers (described above) were further used for PCR. The temperature and cycle conditions were such that after heat denaturation at 94 ℃ for 10 minutes, 25 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 1 minute were carried out.

FVIII F: (above-mentioned)

FVIII R: (above-mentioned)

GAPDH F: (above-mentioned)

GAPDH R: (above-mentioned)

As a result, the expression was equivalent for CHO (FVIIIx1-MAC) and CHO (FVIIIx1-HAC), and higher for CHO (FVIIIx16-MAC) than for CHO (FVIIIx1-MAC) and CHO (FVIIIx 1-HAC).

[C] Gene function analysis of FVIII Gene in CHO cells

To examine whether FVIII protein expression in CHO (FVIIIx1-MAC)1-3 and CHO (FVIIIx1-HAC)1-2 confirmed FVIII mRNA expression as described above was functional, an agglutination assay (Cosmobio) was performed according to the protocol labeled. The cells cultured to 25PDL by 0PDL and non-selective culture were cultured in 6-well dishes and replaced with a new medium from a 100% confluent state. Culture supernatants were recovered after 24 hours and assayed for the degree of FVIII activation due to FVIII activity. As a result, almost no difference in activity was observed between CHO (FVIIIx1-MAC)1-3 and CHO (FVIIIx1-HAC)1-2 in 0 PDL. On the other hand, the activity increased 1.8-fold in CHO (FVIIIx1-MAC)1-3 even in 25PDL, while the activity decreased 1/6-fold in 25PDL in CHO (FVIIIx1-HAC)1-2 (FIG. 67). In addition, the activity of CHO (FVIIIx16-MAC)16-1, 16-2, and 16-3 was increased about 10-fold compared to CHO (FVIIIx1-MAC)1-3, and it was confirmed that the activity was increased depending on the copy number (FIG. 68).

From the above experiments, it was confirmed that functional expression of FVIII gene was observed by carrying F VIII gene on mouse artificial chromosome MAC2 vector, and further functional expression was stably exhibited for a long period of time in FVIII-MAC-carrying CHO as compared with FVIII-HAC-carrying CHO. In addition, DNA encoding a useful protein within 200kb can be inserted by the PAC vector.

Example 15 construction of mouse Artificial chromosome vector MI-MAC into which multiple genes can be introduced

As an example of the possibility of carrying a plurality of genes in the mouse artificial chromosome vector MAC2, a multi-integrase platform having 5 site-specific recombinase recognition sites (. PHI.1attP, R.4attP, TP901-1attP, Bxb1attP, and FRT) was inserted using the Cre/loxP system, and the introduction and expression of a plurality of genes were examined.

[ A.1] production of Multi-Gene-Loading site (Multi-integrase platform) cassette

A cassette having multiple gene-carrying sites for introducing multiple genes into a mouse artificial chromosome vector was prepared as follows using a Multisite-Gateway kit (Invitrogen). First, using PGK-hyg (Clontech) as a template, a gene introduction site, i.e., a Φ C31attP site, an R4attP site, a TP901-1attP site, a Bxb1attP site, and an FRT site, was added to the PGK promoter sequence by the first PCR (each primer pair F1-R1). For PCR, Gene Amp9600 manufactured by Perkin-Elmer, kodpus (Toyobo) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after heat denaturation at 94 ℃ for 2 minutes, 20 cycles of 94 ℃ for 15 seconds, 68 ℃ for 30 seconds and 72 ℃ for 90 seconds were carried out. The PCR product was treated with proteinase K (Gibco) and then purified using CHROMASPIN-TE400 (Clontech).

B1-FRT-PGK-ΦC31 attP-B5rF1：

5'-GAAGTTCCTATACTTTCTAGAGAATAGGAACTTCATTCTACCGGG TAGGGGAGGCGCTTTTCCC-3' (Serial number 111)

B1-FRT-PGK-ΦC31attP-B5rR1：

5'-CAACTGAGAGAACTCAAAGGTTACCCCAGTTGGGGCACTACGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3' (Serial number 112)

B 5-PGK-R4 attP-B4F1：

5'-GGGGACAACTTTGTATACAAAAGTTGATATTCTACCGGGTAGGGGAGGCGCTTTTCCC-3' (Serial number 113)

B5-PGK-R4 attP-B4R1：

5'-CACAAGCAGTACCACTGCTTCAAGTGGTATCGCTTTGGGGAACATGCGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3' (Serial number 114)

B4r-PGK-TP901 attP-B3rF1：

5'-GGGGACAACTTTTCTATACAAAGTTGATATTCTACCGGGTAGGGGAGGCGCTTTTCCC-3' (Serial number 115)

B4r-PGK-TP901 attP-B3rR1：

5'-CTTAATTGAAATAAACGAAATAAAAACTCGCAATTAAGCGAGTTGGAAGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3' (Serial number 116)

B3-PGK-Bxb1 attP-B2F 1：

5'-GGGGACAACTTTGTATAATAAAGTTGGTATTCTACCGGGTAGGGGAGGCGCTTTTCCC-3' (Serial number 117)

B3-PGK-Bxb1 attP-B2R1：

5'-AGACCGCGGTGGTTGACCAGACAAACCACGAAGACACAGGTCATCACGGCCATAGGTCGAAAGGCCCGGAGATGAGGAAGAGGA-3' (Serial number 118)

A second PCR (each primer set F1-R2, F2-R2 only under. PHI. 31) was performed by adding a necessary entry (Gateway) attB sequence to the Multisite-Gateway BP reaction using the first PCR fragment obtained as described above as a template. The same as above is true for PCR conditions and the like, except that the number of cycles is 25 cycles. The sequence of primer F1 was also as described above.

B1-FRT-PGK-ΦC31 attP-B5rF2：

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGGAAGTTCCTATACTTTCTAGAGAATAGGAA-3' (Serial number 119)

B1-FRT-PGK-ΦC31 attP-B5rR2：

5'-GGGGACAACTTTTGTATACAAAGTTGTGACCCTACGCCCCCAACTGAGAGAACTCAAAGGTTACCCCAGT-3' (Serial number 120)

B5-PGK-R4 attP-B4R2：

5'-GGGGACAACTTTGTATAGAAAAGTTGGGTGCACCCGCAGAGTGTACCCACAAGCAGTACCACTGCTTCAAGTGGTAT-3' (Serial number 121)

B4r-PGK-TP901 attP-B3rR2：

5'-GGGGACAACTTTATTATACAAAGTTGTTAAAAGGAGTTTTTTAGTTACCTTAATTGAAATAAACGAAATAAAAACTCG-3' (Serial number 122)

B3-PGK-Bxb1 attP-B2R2：

5'-GGGGACCACTTTGTACAAGAAAGCTGGGTATGGGTTTGTACCGTACACCACTGAGACCGCGGTGGTTGACCAGACAAACCACG-3' (Serial number 123)

Donor vectors (Invitrogen: pDONR221P1-P5R, pDONR221P 5-P4, pDONR221P 4R-P3R, pDONR221P 3-P2) having entry P sequences corresponding to these PCR fragments were mixed, and entry vectors (pENTR L1-FRT-PGK-PhiC 31-R5, pENTRL5-PGK-R4-L4, pENTR 4-PGK-TP901-1-R3, pENTR L3-PGK-Bxb1-L2) were prepared by an in vitro recombination reaction (BP reaction) using BPclonase (FIG. 69). The BP reaction was carried out according to the recommended conditions.

Next, a plasmid having a 3' HPRT-loxP site inserted therein, which is necessary for introducing these gene introduction sites into the mouse artificial chromosome vector, was prepared by the following procedure. Amplification was performed using the following primers using X3.1 as a template. The PCR conditions were the same as those described above (cycle number 25).

PGK 2362: 5'-TGATTGTTCAGGAGGAGGAAGCCGGTGGCG-3' (Serial number 124)

loxP 4548: 5'-AGAGCCTTCAACCCAGTCAGCTCCTTCGAA-3' (Serial number 125)

The PCR fragment was blunt-ended with DEST cassette (Invitrogen: R1-ccdB-Cm-R2) as pDEST. Subsequently, this pDEST was mixed with the above-prepared entry vectors (pENTRL 1-FRT-PGK-. phi.C 31-R5, pENTR L5-PGK-R4-L4, pENTRR4-PGK-TP901-1-R3, pENTR L3-PGK-Bxb1-L2) to prepare a multiple gene-loading site (multiple integrase platform) cassette by an in vitro recombination reaction (LR reaction) using LRclonase (FIG. 70). The LR reaction was carried out according to the recommended conditions.

[ A.2] Loading of multiple Gene-Loading site (multiple integrase platform) cassette into mouse Artificial chromosome vector

The multiple Gene-Loading site (multiple integrase platform) cassette was ligated to the CHO (HPRT) described above^-(ii) a MAC2) and CHO (HPRT) described below^-(ii) a MAC4) after Cre-loxP recombination as described above, HAT-resistant clones were obtained and inserted into mouse artificial chromosome vectors MAC2 or MAC4 (referred to as MI-MAC) (fig. 71).

[ A.3] preparation of Gene transfer cassette

Cassette vectors for introducing foreign genes into the multiple integrase platform were prepared as follows. First, a promoter-free neomycin-resistant gene, which is necessary for drug selection, was amplified using pIRES Neo2(Clontech) as a template and the following primers. The PCR conditions were the same as those described above (cycle number 25).

And (3) NeoF: 5'-AAAGATATCAACTCGAGATGGGATCGGCCATTGAACAAGATGGATTG-3' (Serial number 126)

And (3) NeoR: 5'-TTTGCTAGCCCCCAGCTGGTTCTTTCCGCCTCAGAAGCC-3' (Serial number 127)

Then, this PCR fragment was blunt-ended cloned on SLR assay (Toyobo) after cleavage with restriction enzymes EcoRV and SmaI sites to prepare pNeo. Next, recombination sequences corresponding to each attP site or FRT site (. PHI.C 31attB, R4attB, TP901-1 attB, Bxb 1attB, FRT) were resynthesized (PHI.C 31, Bxb1, FRT: Integrated DNAtechnologies Inc., R4, TP 901-1: Invitrogen). pNeo was cleaved with restriction enzyme SalI, and a DNA fragment containing Φ C31attB or R4attB was excised from the vector synthesized above with restriction enzyme SalI and ligated (pNeo- Φ C31attB, pNeo-R4 attB). Similarly, pNeo was cleaved with restriction enzyme ClaI, a DNA fragment containing TP901-1 attB or FRT was excised from the vector synthesized above with restriction enzyme ClaI and ligated to prepare pNeo-TP901-1 attB or pNeo-FRT, or pNeo was cleaved with restriction enzyme NheI, and a DNA fragment containing Bxb 1attB was excised from the vector synthesized above with restriction enzyme NheI and ligated to prepare pNeo-Bxb1 attB. These vectors can be used to insert any foreign gene into the BamHI site, and then they can be used to form cassette vectors which can be mounted on mouse artificial chromosomes having multiple gene-carrying sites (FIG. 72).

[ A.4] preparation of expression vector for site-specific recombinase

Site-specific recombinases (Φ C31 integrase, R4 integrase, TP901-1 integrase, Bxb1 integrase) which lead to recombination between the respective corresponding attB-attP (GenBank accession No.: Φ C31, CAA 07153; R4, BAA 07372; TP901-1, CAA 59475; Bxb1, AAG59740) were resynthesized (Φ C31: Codon device, others: Invitrogen). For high expression in mammalian cells, codon usage is optimized in mammals to synthesize these integrases. From the synthesized vector, a DNA fragment containing Φ C31 integrase was excised with restriction enzymes KpnI-XbaI and ligated with pVAX1(Invitrogen) excised with restriction enzymes KpnI-XbaI to prepare pCMV- Φ C31, or a DNA fragment containing R4 integrase or TP901-1 integrase and Bxb1 integrase was excised with restriction enzymes NheI-XhoI and ligated with pVAX1(Invitrogen) excised with restriction enzymes NheI-XhoI to prepare pCMV-R4, pCMV-TP901-1 and pCMV-Bxb1 (FIG. 72).

[ A.5] Gene transfer into MI-MAC vector

By introducing the various site-specific recombinase expression vectors described above instead of the Cre expression vector and introducing the gene introduction cassette instead of the FVIII insertion vector, a plurality of (1 to 5) genes can be inserted into the mouse artificial chromosome vector MI-MAC vector. Further, since a plurality of multigene-carrying site (multiintegrase platform) cassettes may be carried on the mouse artificial chromosome MAC vector, the gene may be inserted without limitation (fig. 72).

EXAMPLE 16 construction of mouse Artificial chromosome vector PXR-MAC

A PXR-MAC is constructed by inserting human PXR serving as a nuclear receptor into a mouse artificial chromosome vector MAC3 by using a Cre/loxP system.

[ A.1] preparation of human PXR insertion vector

The basic BAC vector for inserting the human PXR gene and loxP sequence uses RP11-169N13(CHORI) containing the total length of the human PXR gene. According to the method of Yamada et al (J Hum Genet.2008; 53 (5): 447-53), an Amp-5' HPRT-loxP sequence (vector name: PXR-loxP) for insertion into mouse artificial chromosome vector MAC3 was inserted into the kanamycin-resistant gene region on the above BAC vector by homologous recombination.

The site specific DNA generated by human PXR insertion using the HPRT reconstruction system is shown inserted using the Cre/loxP system (fig. 73).

[ A.2] transfection and isolation of HAT-resistant clones

Gene transfer was carried out by lipofection, and Cre 1. mu.g and PXR-loxP vector 2. mu.g were introduced into 6-well cells that reached 90% confluence according to the commercially available protocol (Invitrogen). When 2 weeks of culture were carried out under HAT selective culture, resistant colonies appeared, and a total of 11 colonies obtained by 2 introductions were separated and proliferated for subsequent analysis (clone name: CHO (PXR-MAC)).

[ A.3] screening of drug-resistant clones

[ A.3.1] PCR analysis

In order to screen for recombinants using genomic DNA of HAT-resistant strains as templates, PCR was performed using the following primers to confirm whether or not the PXR gene was site-specifically inserted. The primer sequences are shown below.

TRANS L1 (supra)

TRANS R1 (supra)

hPXR 1L: 5'-aaacagcaaggcaagcatcca-3' (Serial number 128)

hPXR 1R: 5'-tgctttaatccagccctggtg-3' (Serial number 129)

hPXR 2L: 5'-tgtttgctcaatcgtggtctcc-3' (Serial number 130)

hPXR 2R: 5'-acaaaagccgaatgtggtgga-3' (Serial number 131)

hPXR 3L: 5'-ccaagaggcccagaagcaaa-3' (Serial number 132)

hPXR 3R: 5'-tccccacatacacggcagatt-3' (Serial number 133)

hPXR 4L: 5'-acactgccaagagccgacaat-3' (Serial number 134)

hPXR 4R: 5'-gcaaccttgcctctctgatggt-3' (Serial number 135)

hPXR 5L: 5'-tcaaggtgtggaagggaccaa-3' (Serial number 136)

hPXR 5R: 5'-acaaagcagctcggaagagga-3' (Serial number 137)

hPXR 6L: 5'-gttgtcctggggctggaat-3' (Serial number 138)

hPXR 6R: 5'-caaggcaggcactttcataccc-3' (Serial number 139)

kj neo (above)

m 116R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer was used as a thermal cycler, Ampli Taq Gold (Applied Biosystems) was used as a Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. After heat denaturation at 94 ℃ for 10 minutes, 35 cycles of 94 ℃ for 30 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds were carried out. As a result of PCR, 6 clones out of 11 clones were positive for all primers, and the 6 clones were used for subsequent analyses.

[ A.3.3] two-color FISH analysis

Among the 6 clones selected from the results obtained above, two-color FISH analysis was performed according to Songen et al (FISH protocol, Xiu run Co., 1994). FISH analysis was performed using mouse cot-1DNA and human PXR-BAC-derived DNA (RP11-169N13) (CHORI) as probes, and it was confirmed that 5 out of 6 clones carried PXR-MAC at a rate of 60% or more and further showed a PXR-BAC-derived signal, and no signal was detected on MAC3 before site-specific insertion of PXR-BAC as a negative control, and thus, a human PXR gene was site-specifically inserted (FIG. 74).

The above experiment confirmed that a CHO cell carrying mouse artificial chromosome vector PXR-MAC was obtained by carrying human PXR gene on mouse artificial chromosome MAC 3.

[B] Introduction of mouse Artificial chromosome vector PXR-MAC into mouse ES cells from CHO cells containing mouse Artificial chromosome vector PXR-MAC

To prepare chimeric mice carrying PXR-MAC, the chimeric mice were prepared by micronuclear cell fusion from [ A ] as described above]The obtained CHO cells carrying PXR-MAC were introduced into mouse ES cells (wild type TT 2F). According to the method of Tomizuka et al (NatureGenet.16: 133, 1997), approximately 10⁸CHO cells (CHO (PXR-MAC)7, 9, 10, etc.) carrying PXR-MAC were purified minicells and suspended in 5ml of DMEM. About 10 by trypsin treatment⁷Mouse ES cells TT2F were washed 3 times with DMEM, suspended in 5ml of DMEM, added to the centrifuged minicells, and centrifuged at 1250rpm for 10 minutes to completely remove the supernatant. The pellet was sufficiently disentangled by gently beating, and 1: 1.4PEG solution [ 5g of PEG1000 (Wako pure chemical industries, Ltd.), 1ml of DMSO (Sigma) ] was added to 6ml of DMEM]0.5ml, and stirred well for about 1 minute and 30 seconds. Then, 10ml of DMEM was slowly added thereto, centrifuged at 1250rpm for 10 minutes, suspended in 30ml of ES medium, and poured into 3 petri dishes (cones) of 100mm in diameter, which were previously inoculated with feeder cells, to culture the cells. After 24 hours, the medium was replaced with a medium containing G418 at a concentration of 300. mu.g/ml, and selection culture was carried out for about 1 week. As a result, a total of 34 colonies were separated and proliferated for subsequent analysis. 2 clones from CHO (PXR-MAC)7, 2 clones from CHO (PXR-MAC)9, 12 clones from CHO (PXR-MAC)10 were positive in PCR using the above primers that detected only the PXR-MAC region. Furthermore, for the above 16 clones, human-derived clones were usedPXR-BAC DNA (RP11-169N13) (CHORI) was subjected to FISH analysis (Tomizuka et al, Nature Genet.16: 133, 1997), and as a result, the clones specifically detected using the above-described probe were 4 out of 16 clones. From the above, the following conclusions can be drawn: TT2F cells carrying PXR-MAC were obtained from 4 clones (FIG. 75).

[C] As described in example 8, chimeric mice were prepared from mouse ES cells carrying the mouse artificial chromosome vector PXR-MAC, and mouse system TC (PXR-MAC) in which PXR-MAC was transferred as progeny was prepared. In addition, the TC (PXR-MAC) mouse system described above can be used to test the stability of PXR-MAC in somatic cells. In addition, TC (PXR-MAC) mouse system can reproduce CYP gene expression induction by using medicine in human. By mating with the above TC (CYP3A-MAC) mouse system, a TC (CYP3A-MAC/PXR-MAC) system can be prepared, and the obtained product can be used as a model mouse for in vivo tests for investigating drug efficacy and toxicity in the development of pharmaceuticals.

EXAMPLE 17 construction of mouse Artificial chromosome vector MAC4

Mouse artificial chromosome vector MAC4 in which GFP-5' HPRT-loxP-PGKhyg type loxP sequence as DNA insertion sequence was inserted into mouse artificial chromosome MAC was constructed. The 5' HPRT-loxP-PGKhyg type loxP site was inserted into the 21 chromosome-derived GFP-carrying HAC vector (21HAC2) described in Kazuki et al report (Gene therapy: PMID: 21085194, 2010), and the gene expression of HAC and MAC was compared using the same vector. The gene transfer vector for insertion into 21HAC2 can be used as it is without a step of vector preparation.

[A] Insertion of GFP-5' HPRT-loxP-hyg type loxP sequence into mouse artificial chromosome MAC

[ A.1] preparation of GFP-5' HPRT-loxP-hyg type loxP targeting vector

The basic plasmid for loxP site insertion was pMAC2 prepared as described above. HS4-CAG-EGFP-HS4 (obtained by doctor Okagaku, Osaka university and Bo. Felsenfeld, NIH) excised by NotI and SalI was smoothed, and pMAC2 was cloned after XhoI cleavage and smoothing (vector name: pMAC 4). The targeting vector, the target sequence, and the chromosomal allele produced by homologous recombination are shown in FIG. 76.

[ A.2] transfection and isolation of drug-resistant clones

The targeting vector pMAC4 prepared above was linearized with restriction enzyme NotI (TAKARA) in the same manner as described above, transfected into the clone DT40(MAC) prepared above, replaced with a hygromycin (1.5mg/ml) containing medium, and then injected into 2 96-well plates for about 2 weeks of selective culture. A total of 36 resistant colonies obtained by 1 transfection were separated and propagated for subsequent analysis (clone name: DT40(MAC4))

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

In order to extract genomic DNA of hygromycin-resistant strains as templates and select recombinants, PCR was performed using the following primers to confirm whether recombination occurred site-specifically at the position of the mouse chromosomal vector MAC. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: 5'-AGATCTCGGCTAGAGGTACCCTAGAAGATC-3' (Serial number 140)

hygF (244): (above-mentioned)

m 116R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 7 minutes at 68 ℃ were carried out. As a result of PCR, 5 clones out of 36 clones were positive for all primer sets, and therefore, the 5 clones were used for subsequent analysis.

[ A.3.2] two-color FISH analysis

Two-color FISH analysis was performed on 5 clones of DT40(MAC4) obtained as described above according to Songen et al (FISH protocol, Xiu run Co., 1994). In contrast to the results of FISH analysis using mouse cot-1DNA and GFP-5' HPRT-loxP-hyg cassette as probes, no signal derived from the probes was detected in the pre-targeting mouse artificial chromosome vector MAC as a negative control, and in 5 clones of DT40(MAC4), the signal derived from the probes was detected at a rate of 65% or more, and thus site-specifically induced recombination was visually confirmed in the above 5 clones (FIG. 77). From these results, the following conclusions can be drawn: DT40 cell clones carrying the mouse artificial chromosome vector MAC4 were obtained.

[B] Introduction of MAC4 from Chicken DT40 cells containing mouse Artificial chromosome vector MAC4 into CHO cells

[ B.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) cells (accession number JCRB0218, obtained from the resource pool of health scientific research in Japan) that are CHOhprt deficient cells were treated with DT40(MAC4) -B1-5 and B1-74 as recipient cells, and B2-3 and B2-4 in the same manner as described above^-) The micronucleus cell fusion method was performed. A total of 23 resistant colonies obtained by 4 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: CHO (HPRT)^-；MAC4))

[ B.2] screening of drug-resistant clones

[ B.2.1] PCR analysis

In order to extract genomic DNA of hygromycin-resistant strains as a template for selection of recombinants, PCR was performed using the following primers to confirm whether mouse artificial chromosome vector MAC4 could be introduced into CHO cells. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: (above-mentioned)

hygF (244): (above-mentioned)

m 116R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 7 clones out of the 23 clones were positive for all the primer sets, and the 7 clones were used for subsequent analysis.

[ B.2.2] Monochromatic FISH analysis

The CHO (HPRT) obtained above was subjected to the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC4) was subjected to FISH analysis using mouse Cot-1DNA as a probe, and it was confirmed that 4 clones out of the 7 clones introduced MAC4 into CHO cells at a rate of 95% or more (fig. 78).

From the above results, the following conclusions can be drawn: the mouse artificial chromosome vector MAC4 can be introduced into CHO cells.

[C] As described in example 8, mouse ES cells carrying mouse artificial chromosome vector MAC4 were prepared, and the ES cells were used to examine stability in vitro. Furthermore, chimeric mice were prepared from the ES cells, and mouse system TC (MAC4) in which MAC4 was transmitted as progeny was prepared. In addition, the stability of MAC4 in somatic cells can be tested using the TC (MAC4) mouse system described above.

EXAMPLE 18 construction of mouse Artificial chromosome vector UGT2-MAC

UGT2 cluster as a gene group of human drug metabolizing enzymes was cloned transposition of mouse artificial chromosome vector MAC4 using Cre/loxP system, and UGT2-MAC was constructed in the same manner as in example 3. In addition, UGT2-MAC was tested for stability in mouse ES cells.

[A] Site-specific cleavage in AC125239 on human chromosome 4

To delete the distal gene from the UGT2 gene cluster of human chromosome 4, a site-specific chromosome deletion, telomere truncation, was performed.

[ A.1] preparation of targeting vector pTELpuro-UGT2

A targeting vector ptelupro-UGT 2 for inserting a human telomere sequence in the AC125239 region located near the UGT2 locus on human chromosome 4 and telomeric (approximately 150Kb telomere side) was prepared as follows. First, the AC125239 genomic region was amplified by PCR using the following primers.

UGT2tel 4L; 5'-ttctggcaagccttgaagggacaatact-3' (Serial number 141)

UGT2tel 4R; 5'-gcctattttgcctcataacccactgctc-3' (Serial number 142)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400 (Clontech). Then, cleavage was performed with restriction enzymes PstI (ニツポンジ - ン) and BglII (ニツポンジ - ン), and gel filtration was performed with CHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the PstI and BamHI sites of plasmid pTELpuro (Kuroiwa et al, NatureBiotech., 20: 88, 2002). The orientation of the AC125239 genome sequence is centromere → telomere, and the vector with the cloned AC125239 genome segment in the same orientation with the human telomere sequence is used as a target targeting vector pTELpuro-UGT 2. The size of the final long-armed site-specific cleavage construct was 11.9 kb. The targeting vector, the target sequence, and the chromosomal allele produced by homologous recombination are shown in FIG. 79.

[ A.2] transfection and isolation of drug-resistant clones

Chicken DT40 cells carrying human chromosome 4 (clone name: DT40(hCHr4)) were prepared from A9(KM64-4) (Kugoh et al DNA research 1999) carrying human chromosome 4 by the method described in Kazuki et al BBRC 2004. Subsequently, the targeting vector pTELpuro-UGT2 prepared above was linearized with restriction enzyme PstI (ニツポンジ - ン) in the same manner as described above, transfected into the clone DT40(hCHR4)1 prepared above, and the resulting transformant was replaced with a medium containing puromycin (0.3ug/ml), and the resulting transformant was injected into 10 96-well culture plates and subjected to selective culture for about 2 weeks. A total of 96 resistant colonies obtained by 4 transfections were separated and propagated, followed by further analysis (clone name: DT40(hCHr 4-tel)).

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

In order to select a recombinant using genomic DNA of a puromycin-resistant strain as a template, PCR was performed using primers located at the telomere side of the cleavage site or less as a primary selection, and it was confirmed whether or not site-specific cleavage occurred. The primer sequences are shown below.

CSN1S 1-1L; 5'-tttctcctctcaaggaaaacca-3' (Serial number 143)

CSN1S 1-1R; 5'-gccctccatatggcaagaca-3' (Serial number 144)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer was used as a thermal cycler, Ampli Taq Gold (Applied Biosystems) was used as a Taq polymerase, and a buffer and dN TPs (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 95 ℃ for 10 minutes, followed by 30 cycles at 95 ℃ for 20 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds. Next, for 2 clones not detected by the above primers, whether or not the site-specific homologous recombination PCR was caused was confirmed by PCR using the following primers. The sequence is as follows.

UGT2tel 4L; (above-mentioned)

SK 23: 5'-ggccgctctagaactagtggatc-3' (Serial number 145)

UGT2A 1-1L: 5'-tcttctgcatcaagccacatca-3' (Serial number 146)

UGT2A 1-1R: 5'-agccaatgactaccttccattg-3' (Serial number 147)

UGT2A 1-2L: 5'-atcagggagccaccgtagga-3' (Serial number 148)

UGT2A 1-2R: 5'-gcaggcaagttatgccgtga-3' (Serial number 149)

UGT2A 3-1L: 5'-tgcgcccaaacacatggata-3' (Serial number 150)

UGT2A 3-1R: 5'-tggcagaaatgtaggccatga-3' (Serial number 151)

UGT2B 4-1L: 5'-aggctggaagctgggaaacc-3' (Serial number 152)

UGT2B 4-1R: 5'-cctgcatgaaatggatccaaag-3' (Serial number 153)

UGT2B 7-1L: 5'-ccagcaagaaagattgtgatgc-3' (Serial number 154)

UGT2B 7-1R: 5'-ttctaaccatgaactgggtggt-3' (Serial number 155)

UGT2B 11-1L: 5'-gggtttctgctggcctgtgt-3' (Serial number 156)

UGT2B 11-1R: 5'-tctggttttccagcttcaaatg-3' (Serial number 157)

UGT2B 15-1L: 5'-ggtctccttggcatgcacct-3' (Serial number 158)

UGT2B 15-1R: 5'-tgcaatgcttcttttccagttg-3' (Serial number 159)

UGT2B 15-2L: 5'-cagcatggagggttttaaatgg-3' (Serial number 160)

UGT2B 15-2R: 5'-atgttggcgtgctgcatcc-3' (Serial number 161)

UGT2B 28-1L: 5'-catttgaagctggaaaaccaga-3' (Serial number 162)

UGT2B 28-1R: 5'-cctgggtggtaaatctctgaaa-3' (Serial number 163)

Using the above primers, LATaq (Takara Shuzo) was used for PCR, and buffers and dNTPs (dATP, dCTP, dGTP and dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. A band of about 8kb was detected only in 2 clones which site-specifically caused recombination. No band was detected for negative controls DT40, DT40(hCHR4) 1.

[ A.3.2] two-color FISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of 2 clones among the above clones whose recombination was confirmed using human cot-1DNA and puromycin DNA as probes, it was confirmed that human chromosome 4 was not transposed to the host chromosome in all the clones, and a puromycin-derived signal was detected at the end of the human chromosome 4 fragment and cleaved at the desired site, thereby causing site-specific recombination (FIG. 80).

From the above results, the following conclusions can be drawn: in clones DT40(hCHR4-tel)35 and 73, the cleavage was carried out at the distal end of AC125239 which is more telomeric than the UGT2 gene cluster region.

[B] Site-specific insertion of loxP site into AC074378 of human chromosome 4

For transposition insertion into the mouse artificial chromosome vector MAC4 via the loxP sequence, the loxP sequence was inserted into AC074378 at the proximal end of the UGT2 gene cluster of hCHR4-tel in DT40 cells.

[ B.1] preparation of targeting vector pUGT2loxPneo

A targeting vector pUGT2loxPneo for inserting a recognition sequence loxP of Cre recombinase into a region AC074378 located near the UGT2 locus on human chromosome 4 and on the centromeric side (about 300Kb centromeric side) was prepared as follows. First, the AC074378 genome region was amplified by PCR using the following primers.

UGT2loxP 3L: 5'-ggaacaatcccaatcaaaacctcagtgc-3' (Serial number 164)

UGT2loxP 4R: 5'-cgaggattcaagccacatccctaactct-3' (Serial number 165)

The basic plasmid for inserting the loxP site was V907 (Lexiconetics). For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (Takara Shuzo) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that 35 cycles of heat denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 20 seconds and 68 ℃ for 7 minutes were carried out. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400 (Clontech). Then, the cells were digested with restriction enzymes KpnI (ニツポンジ - ン), EcoRI (ニツポンジ - ン) and BglII (ニツポンジ - ン) and gel-filtered with CHROMASPIN-TE1000 (Clontech). The PCR fragment (2.7kb and 2.6kb) was cloned into KpnI or EcoRI and BglII sites of the V907 plasmid (vector name: V907-UGT2HR 2). Next, FRT-pGKneo-FRT was excised from pNT1.1 (donated from the university of Osaka genetic information laboratory center) as the loxP-FRT-pGKneo-FRT-loxP cassette using EcoRI and BamHI, and cloned into the BglII site of the above X3.1 (vector name: X3.1-FRT-pGKneo-FRT). Next, V907-UGT2HR2 was cleaved with restriction enzyme EcoRI, and the DNA fragment containing loxP was excised from X3.1-FRT-pGKneo-FRT with restriction enzyme EcoRI and ligated. The vector in which the loxP site was oriented in the same direction as the cloned genomic fragment of AC074378 was used as the target vector pUGT2 loxPneo. The size of the final loxP insert construct is 11.1 kb. The targeting vector, the target sequence, and the chromosomal allele produced by homologous recombination are shown in FIG. 81.

[ B.2] transfection and isolation of drug-resistant clones

The targeting vector pUGT2loxPneo prepared as described above was linearized with restriction enzymes NotI (TAKARA) and transfected into chicken DT40 cells carrying human chromosome 4 (clone DT40(hCHr4-tel)35, which was replaced with a medium containing neomycin (1.5mg/ml), and the cells were individually plated in 3 96-well culture plates and selectively cultured for about 2 weeks, and a total of 12 resistant colonies obtained by 2 transfections were separated and proliferated for subsequent analyses (clone name: DT40(hCHr 4-tel-loxP)).

[ B.3] screening of homologous recombinants

[ B.3.1] PCR analysis

Genomic DNA was extracted from the neomycin resistant clones using the Puregene DNA Isolation Kit (Gentra System Co.), and homologous recombinants were identified by PCR using the following 2 sets of primers.

Identification of homologous recombinants was performed by PCR using the following 2 sets of primers.

UGT2loxP3L (above)

TRANS R1 (supra)

PGKr1 (supra)

UGT2loxP4R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 4 minutes at 68 ℃ were carried out. 12 clones were screened and 5 clones were identified as homologous recombinants.

[ B.3.2] two-color ISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of 4 clones among the above clones confirmed to be recombined using human cot-1DNA and neomycin DNA as probes, it was confirmed that human chromosome 4 did not transpose to the host chromosome in all clones and a signal derived from neomycin was confirmed in the vicinity of 4q13, and recombination was caused site-specifically (FIG. 82). From the above results, the following conclusions can be drawn: the loxP site as a gene introduction site is site-specifically inserted into AC074378 on human chromosome 4.

[C] hCHr4-loxP-tel introduction from DT40 containing hCHr4-loxP-tel into CHO cells containing MAC 4.

In order to transpose the human UGT2 gene cluster region into the mouse artificial chromosome vector MAC4 via the loxP sequence in CHO cells, hCHR4-loxP-tel was introduced into CHO cells containing the mouse artificial chromosome vector MAC 4.

[ C.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) that is CHOhprt deficient cells (obtained from the Japan health science research resource Bank, accession number JCRB0218) containing MAC4 was treated with DT40 (hCHhr 4-loxP-tel)5 and 10 as recipient cells in the same manner as described above^-(ii) a MAC4) was subjected to the micronucleus fusion method. A total of 22 resistant colonies obtained by 3 times of micronuclear cell fusion were separated and grown for subsequent analysis (clone name: CHO (HPRT)^-；MAC4，hChr4-loxP-tel))。

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of a neomycin-resistant strain as a template for selection of recombinants, PCR was performed using the following primers, and it was confirmed that human chromosome 4 fragment could be introduced into CHO cells containing MAC 4. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: (above-mentioned)

hygF (244): (above-mentioned)

m 116R (supra)

UGT2tel 4L; (above-mentioned)

SK23 (described above)

UGT2A1-1L (supra)

UGT2A1-1R (above)

UGT2A1-2L (supra)

UGT2A1-2R (above)

UGT2A3-1L (supra)

UGT2A3-1R (above)

UGT2B4-1L (supra)

UGT2B4-1R (supra)

UGT2B7-1L (supra)

UGT2B7-1R (supra)

UGT2B11-1L (supra)

UGT2B11-1R (supra)

UGT2B15-1L (supra)

UGT2B15-1R (supra)

UGT2B15-2L (supra)

UGT2B15-2R (above)

UGT2B28-1L (supra)

UGT2B28-1R (supra)

UGT2loxP3L (above)

TRANS R1 (supra)

PGKr1 (supra)

UGT2loxP4R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 5 out of 22 clones were positive in all the primer sets, and the 5 clones were used for subsequent analyses.

[ C.2.2] two-color FISH analysis

The above was verified by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)CHO (HPRT) obtained as described above^-(ii) a MAC4, hCHr4-loxP-tel) was subjected to FISH analysis using mouse Cot-1DNA and human Cot-1DNA as probes, and it was confirmed that 90% or more of MAC1 and hCHr4-loxP-tel were introduced into CHO cells at 1 copy or 2 copies in 1 clone (FIG. 83).

From the above results, the following conclusions can be drawn: hCHr4-loxP-tel can be introduced into CHO cells containing mouse artificial chromosome vector MAC 4.

[D]CHO(HPRT^-(ii) a MAC4, hCHr4-loxP-tel) clone 2Mb site-specific transposition into MAC4 vector at the periphery of the human UGT2 gene cluster region (AC 074378-human UGT2 gene cluster-AC 125239)

To stably maintain 2Mb size DNA, i.e., the human UGT2 gene cluster, in mouse individuals, a transposition was inserted into the mouse artificial chromosome vector MAC4 (fig. 84).

[ D.1] transfection and isolation of HAT-resistant clones

The CHO (HPRT) obtained above was subjected to lipofection^-(ii) a MAC4, hCHr4-loxP-tel)8, and Cre 3. mu.g of 6-well cells that reached 90% confluence were introduced according to the commercially available protocol (Invitrogen). When 2 weeks of culture were carried out under HAT selective culture, resistant colonies appeared, and a total of 6 colonies obtained by 2 introductions were separated and propagated for subsequent analysis (clone name: CHO (UGT2-MAC, hCHR 4-. DELTA.UGT 2)).

[ D.2] screening of drug-resistant clones

[ D.2.1] PCR analysis

In order to extract genomic DNA of the HAT-tolerant strain as a template and screen reciprocal transposition clones, PCR was performed using the following primers to confirm whether chromosomal reciprocal transposition was caused on the human chromosome 4 fragment and MAC 4. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: (above-mentioned)

hygF (244): (above-mentioned)

m 116R (supra)

UGT2tel 4L; (above-mentioned)

SK23 (described above)

UGT2A1-1L (supra)

UGT2A1-1R (above)

UGT2A1-2L (supra)

UGT2A1-2R (above)

UGT2A3-1L (supra)

UGT2A3-1R (above)

UGT2B4-1L (supra)

UGT2B4-1R (supra)

UGT2B7-1L (supra)

UGT2B7-1R (supra)

UGT2B11-1L (supra)

UGT2B11-1R (supra)

UGT2B15-1L (supra)

UGT2B15-1R (supra)

UGT2B15-2L (supra)

UGT2B15-2R (above)

UGT2B28-1L (supra)

UGT2B28-1R (supra)

UGT2loxP3L (above)

TRANS R1 (supra)

PGKr1 (supra)

UGT2loxP4R (supra)

TRANS L1 (supra)

TRANS R1 (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, all of the 6 clones were positive in all of the primer sets, and the 6 clones were used for subsequent analyses.

[ D.2.2] two-color FISH analysis

FISH analysis was performed on the 6 clones of CHO (UGT2-MAC, hCHR 4-. DELTA.UGT 2) obtained as described above by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001) using UGT2-BAC (RP11-643N16) (CHORI) DNA and mouse Cot-1DNA as probes, and it was confirmed that a signal derived from Human UGT2 was observed at a ratio of 50% or more in MAC4 in 2 of the 6 clones (FIG. 85).

From the above results, the following conclusions can be drawn: the 2Mb UGT2 cluster on the human chromosome 4 fragment can be used for cloning the mouse artificial chromosome vector MAC4 by mutual transposition.

[E] Transfer of UGT2-MAC from CHO cells into mouse A9 cells

To prepare mouse ES cells carrying UGT2-MAC, CHO cells carrying UGT2-MAC (CHO (UGT2-MAC, hCHR 4-. DELTA.UGT 2)4, 5) obtained in [ D ] above were introduced into mouse A9 cells having a high minicell-forming ability with respect to mouse A9 cells by micronucleus fusion method. A total of 16 resistant colonies obtained by 4 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: A9(UGT 2-MAC)). As a result, 5 clones were positive in PCR using the above primers for detecting only UGT2-MAC region. Furthermore, FISH analysis was performed using UGT2-BAC (RP11-643N16) (CHORI) and a mouse secondary satellite DNA probe (Tomizuka et al, Nature Genet.16: 133, 1997), and as a result, the presence of UGT2-MAC specifically detected by the probe was confirmed in all of the 5 clones (FIG. 86). The following conclusions can be drawn as above: a9 cells carrying UGT2-MAC were obtained from 5 clones.

[F] Transfer of UGT2-MAC from A9 cells into mouse ES cells

To prepare chimeric mice carrying UGT2-MAC, the chimeric mice were prepared from [ E ] above by micronuclear cell fusion]The A9 cell carrying UGT2-MAC thus obtained was introduced into a mouse ES cell (wild type TT 2F). According to the method of Tomizuka et al (NatureGenet.16: 133, 1997), from about 10⁸A9 cells (A9(UGT2-MAC)13, 15, etc.) carrying UGT2-MAC were purified and suspended in DMEM 5 ml. About 10 by trypsin treatment⁷Mouse ES cells TT2F were washed 3 times with DMEM, suspended in 5ml of DMEM, added to the centrifuged minicells, and centrifuged at 1250rpm for 10 minutes to completely remove the supernatant. The pellet was sufficiently disentangled by gently beating, and 1: 1.4PEG solution [ 5g of PEG1000 (Wako pure chemical industries, Ltd.), 1ml of DMSO (Sigma) ] was added to 6ml of DMEM]0.5ml, and stirred well for about 1 minute and 30 seconds. Then, 10ml of DMEM was added slowly, the mixture was centrifuged at 1250rpm for 10 minutes, and the supernatant was suspended in 30ml of ES medium and poured into 3 petri dishes (Cone Co., Ltd.) with a diameter of 100mm, on which feeder cells were previously seeded, to culture the cells. After 24 hours, the medium was replaced with a medium containing G418 at a concentration of 300. mu.g/ml, and selection culture was carried out for about 1 week. As a result, a total of 25 colonies were separated and proliferated for subsequent analysis. 5 clones from A9(UGT2-MAC)13, 4 clones from A9(UGT2-MAC)15 were positive in PCR using the above primers detecting only the UGT2-MAC region. Furthermore, FISH analysis was performed on the above clones using UGT2-BAC (RP11-643N16) (CHORI) and mouse minor satellite DNA (Tomizuka et al, Nature Genet.16: 133, 1997), and as a result, the above probes were specifically detected and the normal karyotype of the mouse was found to be 7 clones (FIG. 87). The following conclusions can be drawn as above: TT2F cells carrying UGT2-MAC were obtained from 7 clones.

[G] Stability in mouse ES cells of UGT2-MAC

The mouse ES clones obtained as described above (for example, TT2F (UGT2-MAC)9, 10, and 19, obtained in [ F ]) were tested for the proportion of UGT 2-MAC-carrying cells obtained by FISH analysis after long-term culture in non-selective culture in 0 to 50PDL, and found to have a carrying rate of 95% or more even in 50 PDL. (FIG. 88).

[H] As described in example 8, chimeric mice were prepared from mouse ES cells carrying the mouse artificial chromosome vector UGT2-MAC, and mouse system TC (UGT2-MAC) in which UGT2-MAC was transferred as progeny was prepared. In addition, the stability of UGT2-MAC in somatic cells can be tested using the TC (UGT2-MAC) mouse system described above. Since the TC (UGT2-MAC) mouse system can reproduce drug metabolism in humans, it is useful as a model mouse for in vivo tests for examining the efficacy and toxicity of the second phase reaction in drug development.

EXAMPLE 19 construction of mouse Artificial chromosome vector CYP2C-MAC

CYP2C cluster, which is a gene group of human drug metabolizing enzymes, was cloned by transposition of mouse artificial chromosome vector MAC4 using Cre/loxP system, and CYP2C-MAC was constructed in the same manner as in example 3.

[A] Site-specific cleavage in AL157834 on human chromosome 10

In order to delete the distal gene from the CYP2C gene cluster of human chromosome 10, site-specific chromosome deletion, i.e., telomere truncation, was performed.

[ A.1] preparation of targeting vector pTELpuro-CYP2C

A targeting vector pTELpuro-CYP2C for inserting a human telomere sequence into the AL157834 region located near the CYP2C locus on human chromosome 10 and on the telomere side (approximately 150Kb telomere side) was prepared as follows. First, the AL157834 genomic region was amplified by PCR using the following primers.

2Ctel 2L; 5'-GCTATGAGACACAGGGCAGCTGAAAGTC-3' (Serial number 166)

2Ctel 2R; 5'-TTGTGAACCACCATGCCTAGCTGAAAGT-3' (Serial number 167)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400 (Clontech). Then, it was cut with restriction enzymes BamHI (ニツポンジ - ン) and BglII (ニツポンジ - ン) and gel-filtered with CHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the BamHI site of the plasmid pTELpuro (Kuroiwa et al, NatureBiotech., 20: 88, 2002). The orientation of AL157834 genome sequence is centromere → telomere, and the cloned AL157834 genome fragment and human telomere sequence are in the same orientation as the target targeting vector pTELpuro-CYP 2C. The size of the final long-arm proximal site-specific cleavage construct was 11.6 kb. The targeting vector, the target sequence, and the chromosomal allele produced by homologous recombination are shown in FIG. 89.

[ A.2] transfection and isolation of drug-resistant clones

Chicken DT40 cells carrying human chromosome 10 (clone name: DT40(hCHr10)) were prepared from A9(KM32-2) and A9(KM26-3) carrying human chromosome 10 (DNA research 1999, Kugoh et al) by the method described in Kazuki et al BBRC 2004. Subsequently, the targeting vector pTELpuro-CYP2C prepared above was linearized with restriction enzyme PstI (ニツポンジ - ン) in the same manner as described above, transfected into the clones DT40(hCHR10)1 and 42 prepared above, and the resulting clones were replaced with puromycin (0.3ug/ml) containing medium, and the resulting clones were individually plated into 10 96-well culture plates and subjected to selective culture for about 2 weeks. A total of 96 resistant colonies obtained by 4 transfections were separated and propagated, followed by further analysis (clone name: DT40(hCHr 10-tel)).

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

In order to select a recombinant using genomic DNA of a puromycin-resistant strain as a template, PCR was performed using primers located at the telomere side of the cleavage site or less as a primary selection, and it was confirmed whether or not site-specific cleavage occurred. The primer sequences are shown below.

h10yi _ 1F; 5'-ACGGGGCTCCTACTCTTGTC-3' (Serial number 168)

h10yi _ 1R; 5'-GCTTCCACCTGCATCTCAC-3' (Serial number 169)

h10yi _ 2F; 5'-CAATGCCTTATGCATGTTGTG-3' (Serial number 170)

h10yi _ 2R; 5'-TCCACAGCATACTGCTGACC-3' (Serial number 171)

h10yi _ 3F; 5'-AAGGAAGGTGACCGCCTACT-3' (Serial number 172)

h10yi _ 3R; 5'-CATCCGAGGACATCTTTGGT-3' (Serial number 173)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer was used as a thermal cycler, Ampli Taq Gold (Applied Biosystems) was used as a Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 95 ℃ for 10 minutes, followed by 30 cycles at 95 ℃ for 20 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds. Subsequently, it was confirmed by PCR using the following primers whether or not site-specific homologous recombination was caused on 3 clones not detected by the above primers. The sequence is as follows.

2Ctel 4L; 5'-ATCTGCAGGGAAGGGATCCAGTTTCAGCTTCCTAC-3' (Serial number 174)

SK23 (described above)

CYP2C 8-1F: 5'-ACATGTCAAAGAGACACACA-3' (Serial number 175)

CYP2C 8-1R: 5'-TAGCATATTTCCAATAATAGGA-3' (Serial number 176)

CYP2C 9-1F: 5'-AGAAGGCTTCAATGGATTCTC-3' (Serial number 177)

CYP2C 9-1R: 5'-TGTCCTTAATACCTATCTGTAGG-3' (Serial number 178)

CYP2C 18-1F: 5'-ACAGCTGGATCCATTGAAGG-3' (Serial number 179)

CYP2C 19-1F: 5'-ACACACACTTAATTAGCATGGA-3' (Serial number 180)

CYP2C 19-1R: 5'-TTGGTTAAGGATTTGCTGACA-3' (Serial number 181)

Using the above primers, LATaq (Takara Shuzo) was used for PCR, and buffers and dNTPs (dATP, dCTP, dGTP and dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. A band of about 8kb was detected only in 2 clones which site-specifically caused recombination. No bands were detected for negative controls DT40, DT40(hCHR10)1, 42.

[ B.3.2] two-color FISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of the 3 clones confirmed to be recombined using human cot-1DNA and puromycin DNA as probes, it was confirmed that human chromosome 10 was not transposed to the host chromosome in all the clones, and a puromycin-derived signal was detected at the end of the human chromosome 10 fragment and cleaved at the desired site, thereby causing site-specific recombination (FIG. 90).

From the above results, the following conclusions can be drawn: in clones DT40(hCHR10-tel)5 and 98 and 101, the cut was made at the distal end of AL157834, which is more telomeric than the CYP2C gene cluster region.

[B] Site-specific insertion of loxP site into AL138759 of human chromosome 10

To transpose and insert the loxP site into the mouse artificial chromosome vector MAC4 via the loxP site, the loxP site was inserted into AL138759 at the proximal end of CYP2C gene cluster of hCHR10-tel in DT40 cells.

[ B.1] preparation of targeting vector pCYP2 CloxPino

A targeting vector pCYP2CloxPneo for inserting a recognition sequence loxP of Cre recombinase into the AL138759 region located near the CYP2C locus on human chromosome 10 and on the centromere side (about 300Kb centromere side) was prepared as follows. First, the AL138759 genome region was amplified by PCR using the following primers.

hloxP-SacII-EcoRI-F: 5'-TCCCCGCGGATCTGCTCCATACTCTGTACC-3' (Serial number 182)

hloxP-1R: 5'-CATTCAAGGGGTTCTGGGTCTGTAAACT-3' (Serial number 183)

The basic plasmid for inserting the loxP site was V907(Lexicon genetics). For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (Takara Shuzo) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation at 98 ℃ for 20 seconds and 68 ℃ for 7 minutes were carried out. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400 (Clontech). Then, it was cleaved with restriction enzymes SacII (ニツポンジ - ン) and EcoRI (ニツポンジ - ン) and BamHI (ニツポンジ - ン) and gel-filtered with CHROMASPIN-TE1000 (Clontech). The PCR fragments (1.5kb and 3.0kb) were cloned into the SacII and EcoRI or EcoRI and BamHI sites of the V907 plasmid (vector name: V907-CYP2CHR 2). Next, V907-CYP2CHR2 was cleaved with restriction enzyme EcoRI, and the DNA fragment containing loxP was excised from the above X3.1-FRT-pGKneo-FRT with restriction enzyme EcoRI and ligated. The vector with loxP site oriented in the same direction as the cloned AL138759 genome fragment was used as targeting vector pCYP2 CloxPneo. The size of the final loxP insert construct is 10.3 kb. The targeting vector, the target sequence, and the chromosomal allele produced by homologous recombination are shown in FIG. 91.

[ B.2] transfection and isolation of drug-resistant clones

The targeting vector pCYP2CloxPneo prepared above was linearized with restriction enzymes NotI (TAKARA) in the same manner as described above, transfected into chicken DT40 cells carrying human chromosome 10 (clone DT40(hCHr10-tel)1-98, replaced with a medium containing neomycin (1.5mg/ml), individually plated in 3 96-well culture plates, selectively cultured for about 2 weeks, and a total of 15 resistant colonies obtained by 2 transfections were separated and proliferated for subsequent analysis (clone name: DT40(hCHr 10-tel-loxP)).

[ B.3] screening of homologous recombinants

[ B.3.1] PCR analysis

Genomic DNA was extracted from the neomycin resistant clones using the Puregene DNA Isolation Kit (Gentra System Co.), and homologous recombinants were identified by PCR using the following 2 sets of primers.

Identification of homologous recombinants was performed by PCR using the following 2 sets of primers.

hloxP-SacII-EcoRI-F (above)

TRANS R1 (supra)

PGKr1 (above)

hloxP-1R (above)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 4 minutes at 68 ℃ were carried out. Screening of 15 clones resulted in 1 clone being identified as a homologous recombinant.

[ B.3.2] two-color FISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of 1 clone among the above clones confirmed to be recombined using human cot-1DNA and neomycin as probes, it was confirmed that human chromosome 10 was not transposed to the host chromosome in all the clones, and a signal derived from neomycin was detected in the vicinity of 10q24, thereby causing recombination site-specifically. From the above results, the following conclusions can be drawn: the loxP site as a gene introduction site is site-specifically inserted into AL138759 on human chromosome 10.

[C] hCHr10-loxP-tel introduction from DT40 containing hCHr10-loxP-tel into CHO cells containing MAC 4.

In order to transpose the human CYP2C gene cluster region into the mouse artificial chromosome vector MAC4 via the loxP sequence in CHO cells, hCHR10-loxP-tel was introduced into CHO cells containing the mouse artificial chromosome vector MAC 4.

[ C.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) that is CHOhprt deficient cells (obtained from the Japan health science research resource Bank, accession number JCRB0218) containing MAC4 was treated with DT40(hCHr10-loxP-tel)7 as recipient cells in the same manner as described above^-(ii) a MAC4) was subjected to the micronucleus fusion method. A total of 8 resistant colonies obtained by 3 times of micronuclear cell fusion were separated and grown for subsequent analysis (clone name: CHO (HPRT)^-；MAC4，hChr10-loxP-tel))。

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of a neomycin-resistant strain as a template for selection of recombinants, PCR was performed using the following primers to confirm whether or not the human chromosome 10 fragment was introduced into CHO cells containing MAC 4. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: (above-mentioned)

hygF (244): (above-mentioned)

m 116R (supra)

2Ctel4L (supra)

SK23 (described above)

CYP2C8-1F (supra)

CYP2C8-1R (supra)

CYP2C9-1F (supra)

CYP2C9-1R (supra)

CYP2C18-1F (supra)

CYP2C19-1F (supra)

CYP2C19-1R (supra)

hloxP-SacII-EcoRI-F (above)

TRANS R1 (supra)

PGKr1 (supra)

hloxP-1R (above)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, all of the 8 clones were positive in all of the primer sets, and the 8 clones were used for subsequent analyses.

[ C.2.2] two-color FISH analysis

The CHO (HPRT) obtained above was subjected to the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC4, hCHr10-loxP-tel) was subjected to FISH analysis using mouse Cot-1DNA and human Cot-1DNA as probes, and it was confirmed that MAC1 and hCHr10-loxP-tel were introduced into CHO cells at a rate of 90% or more in 2 clones at 1 copy or 2 copies (FIG. 92).

From the above results, the following conclusions can be drawn: hCHr10-loxP-tel can be introduced into CHO cells containing mouse artificial chromosome vector MAC 4.

[D]CHO(HPRT^-(ii) a MAC4, hCHr10-loxP-tel) cloneThe position specificity transposition of 380kb of the human CYP2C gene cluster region (AL 138759-human CYP2C gene cluster-AL 157834) to the MAC4 vector

In order to stably maintain 380kb sized DNA, i.e., the human CYP2C gene cluster, in a mouse individual, a transposition was inserted into the mouse artificial chromosome vector MAC4 (FIG. 93).

[ D.1] transfection and isolation of HAT-resistant clones

The CHO (HPRT) obtained above was subjected to lipofection^-(ii) a MAC4, hCHr10-loxP-tel)1 and 5, and Cre 3. mu.g of 6-well cells that reached 90% confluence were introduced according to the commercially available protocol (Invitrogen). When 2 weeks of culture were carried out under HAT selection culture, resistant colonies appeared, and a total of 11 colonies obtained by 2 introductions were separated and proliferated for subsequent analysis (clone name: CHO (CYP2C-MAC, hCHr 10-. DELTA.CYP 2C)).

[ D.2] screening of drug-resistant clones

[ D.2.1] PCR analysis

In order to extract genomic DNA of the HAT-tolerant strain as a template and screen reciprocal transposition clones, PCR was performed using the following primers to confirm whether chromosomal reciprocal transposition was caused on the human chromosome 10 fragment and MAC 4. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: (above-mentioned)

hygF (244): (above-mentioned)

m 116R (supra)

2Ctel4L (supra)

SK23 (described above)

CYP2C8-1F (supra)

CYP2C8-1R (supra)

CYP2C9-1F (supra)

CYP2C9-1R (supra)

CYP2C18-1F (supra)

CYP2C19-1F (supra)

CYP2C19-1R (supra)

hloxP-SacII-EcoRI-F (above)

PGKr1 (above)

hloxP-1R (above)

TRANS L1 (supra)

TRANS R1 (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 9 out of 11 clones were positive for all the primer sets, and the 9 clones were used for subsequent analyses.

[ D.2.2] two-color FISH analysis

FISH analysis of the 9 clones of CHO (CYP2C-MAC, hCHr 10-. DELTA.CYP 2C) obtained as described above was carried out by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001) using CYP2C-BAC (RP11-466J14) (CHORI) DNA and mouse Cot-1DNA as probes, and it was confirmed that signals derived from Human CYP2C were observed at a ratio of 50% or more in MAC4 of 3 of the 9 clones (FIG. 94).

From the above results, the following conclusions can be drawn: the CYP2C cluster 380kb on the human chromosome 10 fragment can be used for cloning the mouse artificial chromosome vector MAC4 by mutual transposition.

[E] Transfer of CYP2C-MAC from CHO cells into mouse A9 cells

To prepare mouse ES cells carrying CYP2C-MAC, CHO cells carrying CYP2C-MAC (CHO (CYP2C-MAC, hCHr 10-. DELTA.CYP 2C)2, 8, 10) obtained in [ D ] above were introduced into mouse A9 cells having a high minicell-forming ability with respect to mouse A9 cells by the micronucleus fusion method. The total of 4 resistant colonies obtained by 4 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: A9(CYP 2C-MAC)). As a result, 4 clones were positive in PCR using the above primers which detected only the CYP2C-MAC region. Furthermore, FISH analysis was performed using CYP2C-BAC (RP11-466J14) (CHORI) and mouse secondary satellite DNA probes (Tomizuka et al, NatureGenet.16: 133, 1997), and as a result, the presence of CYP2C-MAC specifically detected using the above probes was confirmed in 2 out of 4 clones. The following conclusions can be drawn as above: a9 cells carrying CYP2C-MAC were obtained from 2 clones.

[F] Mouse ES cells carrying the mouse artificial chromosome vector CYP2C-MAC were prepared as described in example 8, and the ES cells were used to examine the stability in vitro. Furthermore, mouse system TC (CYP2C-MAC) in which CYP2C-MAC is transmitted as a progeny can be prepared by directly walking chimeric mice from the ES cells. In addition, the stability of CYP2C-MAC in somatic cells can be tested using the TC (CYP2C-MAC) mouse system described above. Liver microsomes derived from the TC (CYP2C-MAC) mouse system are also useful as samples for drug development to examine the efficacy and toxicity of the first-phase reaction. Since the TC (CYP2C-MAC) mouse system can reproduce drug metabolism in humans, it is useful as a model mouse for in vivo tests for examining drug efficacy and toxicity of the first-phase reaction in drug development.

EXAMPLE 20 construction of mouse Artificial chromosome vector MDR1-MAC

MDR1 gene as a gene group of human drug metabolizing enzymes was cloned by transposition of mouse artificial chromosome vector MAC4 using Cre/loxP system, and MDR1-MAC was constructed in the same manner as in example 3.

[A] Site-specific insertion of loxP site into AC005045 of human chromosome 7

To transpose the vector MAC4 inserted into the mouse artificial chromosome via the loxP sequence, the loxP sequence was inserted into AC005045 proximal to MDR1 gene of human chromosome 7 (hChr7) in DT40 cells.

[ A.1] preparation of targeting vector pMDR1loxPBs

A targeting vector pMDR1loxPBs for inserting a recognition sequence loxP of Cre recombinase into the AC005045 region located near the MDR1 locus on human chromosome 7 and on the centromere side (about 50Kb centromere side) was prepared as follows. First, the AC005045 genomic region was amplified by PCR using the following primers.

MDR1loxP 2L: 5'-gccaagtgtagctggagaatgattcgtg-3' (Serial number 184)

MDR1loxP 1R: 5'-acaaggcacttcaggataccaagcttcc-3' (Serial number 185)

The basic plasmid for inserting the loxP site was V901 (Lexiconetics). For PCR, Gene Amp9600 manufactured by Perkin-Elmer, LATaq (Takara Shuzo) was used as a thermal cycler, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that 35 cycles of heat denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 20 seconds and 68 ℃ for 7 minutes were carried out. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400 (Clontech). Then, it was cleaved with the restriction enzyme BglII (ニツポンジ - ン) and subjected to gel filtration using CHROMASPIN-TE1000 (Clontech). The PCR fragments (2.5kb and 5.3kb) were cloned into the BglII and BamHI sites of the V901 plasmid (vector name: V901-MDR1HR 2). Next, V901-MDR1HR2 was cleaved with restriction enzymes AscI (NEB) and KpnI, and a DNA fragment containing loxP was excised from cassette vector Bs-loxP-3' HPRT (Hoshiya et al, Mol ther. 2009; 17 (2): 309-17) with restriction enzymes AscI and KpnI and ligated. The vector in which the loxP site was oriented in the same direction as the cloned AC005045 genomic fragment was used as the targeting vector pMDR1 loxPBs. The size of the final loxP insert construct is 13.0 kb. The targeting vector, the target sequence, and the chromosomal allele produced by homologous recombination are shown in FIG. 95.

[ A.2] transfection and isolation of drug-resistant clones

The targeting vector pMDR1loxPBs prepared as described above was linearized with restriction enzyme NotI (TAKARA) as described above, transfected into human chromosome 7-carrying chicken DT40 cells (clone DT40- #7) prepared by the method described in WO0I/011951, and replaced with a medium containing blasticidin S (15. mu.g/ml), and 3 96-well culture plates were individually plated and selectively cultured for about 2 weeks. A total of 9 resistant colonies obtained by 2 transfections were separated and propagated for subsequent analysis (clone name: DT40(hCHr 7M-loxP)).

[ A.3] screening for homologous recombinants

[ A.3.1] PCR analysis

Genomic DNA was extracted from the blasticidin S-resistant clones using the Puregene DNA Isolation Kit (Gentra System Co.), and homologous recombinants were identified by PCR using the following 2 sets of primers.

Identification of homologous recombinants was performed by CR using the following 2 sets of primers.

MDR1loxP2L (supra)

BsdR: 5'-gctcaagatgcccctgttct-3' (Serial number 186)

hprt 332F: 5'-aaagatggtcaaggtcgcaa-3' (Serial number 187)

MDR1loxP1R (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃ 35 cycles of 10 seconds at 98 ℃ and 4 minutes at 68 ℃ were carried out. Screening was performed on 9 clones, and as a result, 3 clones were identified as homologous recombinants.

[ A.3.3] two-color FISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of the above-described clone 3in which recombination was confirmed using human cot-1DNA and blasticidin DNA as probes, it was confirmed that human chromosome 7 was not transposed to the host chromosome in all the clones, and a signal derived from neomycin was confirmed in the vicinity of 7q21, and therefore, recombination was caused site-specifically. From the above results, the following conclusions can be drawn: the loxP site as a gene introduction site was site-specifically inserted into AC005045 on human chromosome 7.

[B] Site-specific cleavage in AC003083 on human chromosome 7

To delete the distal gene from the MDR1 gene of human chromosome 7, site-specific chromosome deletion, telomere truncation, was performed.

[ B.1] preparation of targeting vector pTELpuro-MDR1

A targeting vector pTELpuro-MDR1 for inserting a human telomere sequence into an AC003083 region located near the MDR1 locus on human chromosome 7 and telomeric (about 50Kb telomere side) was prepared as follows. First, the AC003083 genomic region was amplified by PCR using the following primers.

MDR1tel 5L; 5'-ctattctaaaaagctgccttggcccaca-3' (Serial number 188)

MDR1tel 5R; 5'-tgtagcccagttcctaatgggacacaga-3' (Serial number 189)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. The PCR product was subjected to protease K (Gibco) treatment and then gel-filtered using CHROMASPIN-TE400 (Clontech). Then, the cells were digested with restriction enzymes EcoRI (ニツポンジ - ン) and PstI (ニツポンジ - ン), and gel-filtered with CHROMASPIN-TE1000 (Clontech). The PCR fragment was cloned into the EcoRI and PstI sites of the plasmid pTELpuro (Kuroiwa et al, NatureBiotech., 20: 88, 2002). The orientation of the AC003083 genome sequence is centromere → telomere, and a vector with the cloned AC003083 genome fragment in the same orientation with the human telomere sequence is used as a target targeting vector pTELpuro-MDR 1. The size of the final long-arm proximal site-specific cleavage construct was 13.1 kb. Targeting vectors, target sequences and chromosomal alleles produced by homologous recombination are shown (FIG. 96).

[ B.2] transfection and isolation of drug-resistant clones

The targeting vector pTELpuro-MDR1 prepared above was linearized with restriction enzyme EcoRI (ニツポンジ - ン) in the same manner as described above, transfected into the clones DT40(hCHR7M-loxP)8 and 9 prepared above, replaced with a medium containing puromycin (0.3ug/ml), and each was injected into 10 96-well culture plates and subjected to selective culture for about 2 weeks. A total of 96 resistant colonies obtained by 4 transfections were separated and propagated, followed by analysis (clone name: DT40(hCHr 7M-loxP-tel)).

[ B.3] screening of homologous recombinants

[ B.3.1] PCR analysis

In order to select a recombinant using genomic DNA of a puromycin-resistant strain as a template, PCR was performed using primers located at the telomere side of the cleavage site or less as a primary selection, and it was confirmed that site-specific cleavage occurred. The primer sequences are shown below.

CYP3A 4R (supra)

CYP3A 4F (supra)

CYP3A 5R (supra)

CYP3A 5F (supra)

CYP3A 7R (supra)

CYP3A 7F (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer was used as a thermal cycler, Ampli Taq Gold (Applied Biosystems) was used as a Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 95 ℃ for 10 minutes, followed by 30 cycles at 95 ℃ for 20 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 30 seconds.

Subsequently, it was confirmed whether site-specific homologous recombination was caused by using the following primer pairs and 3 clones not detected by the above primers. The sequence is as follows.

MDR1tel 5L; 5'-ATCTGCAGGGAAGGGATCCAGTTTCAGCTTCCTAC-3' (Serial number 190)

SK23 (described above)

MDR 1-1L: 5'-ctcctaggagtactcacttc-3' (Serial number 191)

MDR 1-1R: 5'-aacagaaacatggcttggcg-3' (Serial number 192)

MDR 1-2L: 5'-cgccaagccatgtttctgttt-3' (Serial number 193)

MDR 1-2R: 5'-aaggaaatgctttctgccttg-3' (Serial number 194)

MDR 1-3L: 5'-gtgcaacggaagccagaaca-3' (Serial number 195)

MDR 1-3R: 5'-agcggcctctgcttctttga-3' (Serial number 196)

MDR 1-4L: 5'-ctgattggctgggcaggaac-3' (SEQ ID NO. 197)

MDR 1-4R: 5'-cttggaacggccaccaagac-3' (Serial number 198)

MDR 1-5L: 5'-ggtgctggttgctgcttaca-3' (Serial number 199)

MDR 1-5R: 5'-cccaacatcgtgcacatcaa-3' (Serial number 200)

MDR 1-6L: 5'-gtcagtgttgatggacagga-3' (Serial number 201)

MDR 1-6R: 5'-gcattggcttccttgacagc-3' (Serial number 202)

MDR 1-7L: 5'-ggttccaggcttgctgtaat-3' (Serial number 203)

MDR 1-7R: 5'-tctttcagtgcttgtccaga-3' (Serial number 204)

MDR 1-8L: 5'-ggcaaagaaataaagcgactg-3' (Serial number 205)

MDR 1-8R: 5'-cctcctttgctgccctcaca-3' (Serial number 206)

MDR 1-9L: 5'-tcttgtccaaactgcctgtga-3' (Serial number 207)

MDR 1-9R: 5'-tgcaagaatcagcaggatcaa-3' (Serial number 208)

Using the above primers, LATaq (Takara Shuzo) was used for PCR, and buffers and dNTPs (dATP, dCTP, dGTP and dTTP) were used according to the conditions recommended for labeling. The temperature and cycle conditions were such that after 1 minute of thermal denaturation at 94 ℃, 35 cycles of thermal denaturation were carried out at 98 ℃ for 20 seconds and 68 ℃ for 8 minutes. A band of about 8kb was detected only in 3 clones which site-specifically caused recombination. No bands were detected for negative controls DT40, DT40(hCHR7M-loxP)8, 9.

[ B.3.2] two-color FISH analysis

FISH analysis was performed according to Songen et al (FISH protocol, Xiugun Co., 1994). As a result of FISH analysis of the 3 clones confirmed to be recombined using human cot-1DNA and puromycin DNA as probes, it was confirmed that human chromosome 7 was not transposed to the host chromosome in all the clones, and a puromycin-derived signal was detected at the end of the human chromosome 7 fragment and cleaved at the desired site, thereby causing recombination site-specifically (FIG. 97).

From the above results, the following conclusions can be drawn: in clones DT40(hCHr7M-loxP-tel)10, 12 and 70, the cut was made at the distal end of AC003083, which is more telomeric than the MDR1 gene region.

[C] hCHr7M-loxP-tel introduction from DT40 containing hCHr7M-loxP-tel into CHO cells containing MAC 4.

In order to transpose the human MDR1 gene region into the mouse artificial chromosome vector MAC4 via the loxP sequence in CHO cells, hCHr7M-loxP-tel was introduced into CHO cells containing the mouse artificial chromosome vector MAC 4.

[ C.1] isolation of micronuclear cell fusion and drug-resistant clones

CHO (HPRT) that is CHOhprt deficient cells (obtained from the Japan health science research resource Bank, accession number JCRB0218) containing MAC4 was treated with DT40(hCHr7M-loxP-tel)10 and 70 as recipient cells in the same manner as described above^-(ii) a MAC4) was subjected to the micronucleus fusion method. A total of 15 resistant colonies obtained by 4 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: CHO (HPRT)^-；MAC4，hChr7M-loxP-tel))。

[ C.2] screening of drug-resistant clones

[ C.2.1] PCR analysis

In order to extract genomic DNA of the blasticidin S-resistant strain as a template for selection of recombinants, PCR was performed using the following primers to confirm that human chromosome 7 fragment was introduced into CHO cells containing MAC 4. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: (above-mentioned)

hygF (244): (above-mentioned)

m 116R (supra)

MDR1loxP2L (supra)

BsdR: 5'-gctcaagatgcccctgttct-3' (Serial number 209)

hprt 332F: 5'-aaagatggtcaaggtcgcaa-3' (Serial number 210)

MDR1loxP1R (supra)

MDR1tel5L (supra)

SK23 (described above)

MDR1-1L (supra)

MDR1-1R (supra)

MDR1-2L (supra)

MDR1-2R (supra)

MDR1-3L (supra)

MDR1-3R (supra)

MDR1-4L (supra)

MDR1-4R (supra)

MDR1-5L (supra)

MDR1-5R (supra)

MDR1-6L (supra)

MDR1-6R (supra)

MDR1-7L (supra)

MDR1-7R (supra)

MDR1-8L (supra)

MDR1-8R (supra)

MDR1-9L (supra)

MDR1-9R (supra)

As CR, a thermal cycler was used, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 6 clones out of 15 clones were positive in all the primer sets, and the 6 clones were used for subsequent analyses.

[ C.2.2] two-color FISH analysis

The CHO (HPRT) obtained above was subjected to the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001)^-(ii) a MAC4, hCHr7M-loxP-tel) was subjected to F with mouse Cot-1DNA and human Cot-1DNA as probesISH analysis confirmed that MAC1 and hCHr7M-loxP-tel were introduced into CHO cells at a ratio of 80% or more in 2 clones at 1 copy or 2 copy (FIG. 98).

From the above results, the following conclusions can be drawn: hCHr7M-loxP-tel can be introduced into CHO cells containing mouse artificial chromosome vector MAC 4.

[D]CHO(HPRT^-(ii) a MAC4, hCHr7M-loxP-tel) clone 210kb around the human MDR1 gene region (AC 005045-human MDR1 gene-AC 003083) site-specific transposition into MAC4 vector

In order to stably maintain the 210kb size DNA, i.e., the human MDR1 gene, in individual mice, a transposition was inserted into the mouse artificial chromosome vector MAC4 (FIG. 99).

[ D.1] transfection and isolation of HAT-resistant clones

The CHO (HPRT) obtained above was subjected to lipofection^-(ii) a MAC4, hCHr7M-loxP-tel)7 and 15, and Cre 3. mu.g was introduced into 6-well cells that reached 90% confluence according to the commercially available protocol (Invitrogen). When 2 weeks of culture were carried out under HAT selective culture, resistant colonies appeared, and 10 colonies in total obtained by 2 introductions were separated and propagated for subsequent analysis (clone name: CHO (MDR1-MAC, hCHr 7-. DELTA.MDR 1)).

[ D.2] screening of drug-resistant clones

[ D.2.1] PCR analysis

In order to extract genomic DNA of the HAT-tolerant strain as a template and screen reciprocal transposition clones, PCR was performed using the following primers to confirm whether chromosomal reciprocal transposition was caused on the human chromosome 7 fragment and MAC 4. The primer sequences are shown below.

m 114L: (above-mentioned)

V907-NotI-R: (above-mentioned)

hygF (244): (above-mentioned)

m 116R (supra)

MDR1loxP2L (supra)

BsdR: 5'-gctcaagatgcccctgttct-3' (Serial number 211)

hprt 332F: 5'-aaagatggtcaaggtcgcaa-3' (Serial number 212)

MDR1loxP1R (supra)

MDR1tel5L (supra)

SK23 (described above)

MDR1-1L (supra)

MDR1-1R (supra)

MDR1-2L (supra)

MDR1-2R (supra)

MDR1-3L (supra)

MDR1-3R (supra)

MDR1-4L (supra)

MDR1-4R (supra)

MDR1-5L (supra)

MDR1-5R (supra)

MDR1-6L (supra)

MDR1-6R (supra)

MDR1-7L (supra)

MDR1-7R (supra)

MDR1-8L (supra)

MDR1-8R (supra)

MDR1-9L (supra)

MDR1-9R (supra)

TRANSL1 (supra)

TRANSR1 (supra)

For PCR, GeneAmp9600 manufactured by Perkin-Elmer, LATaq (TAKARA) was used as a thermal cycler for Taq polymerase, and a buffer and dNTP (dATP, dCTP, dGTP, dTTP) were used according to the recommended conditions for labeling. The temperature and cycle conditions were such that 30 cycles of thermal denaturation at 94 ℃ for 1 minute and then at 98 ℃ for 10 seconds and 68 ℃ for 7 minutes were carried out. As a result of PCR, 6 out of 10 clones were positive in all the primer sets, and the 6 clones were used for subsequent analyses.

[ D.2.2] two-color FISH analysis

FISH analysis of 6 clones of CHO (MDR1-MAC, hCHR 7-. DELTA.MDR 1) obtained as described above was carried out by the method described in Shinohara et al, report (Human Molecular Genetics, 10: 1163-1175, 2001) using MDR1-BAC (RP11-784L5) (CHORI) DNA and mouse Cot-1DNA as probes, and it was confirmed that signals derived from Human MDR1 were observed at a ratio of 60% or more in MAC4 of 3 clones out of 6 clones (FIG. 100).

From the above results, the following conclusions can be drawn: the MDR1 gene 210kb on the human chromosome 7 fragment can be used for cloning the mouse artificial chromosome vector MAC4 by mutual transposition.

[E] Transfer of MDR1-MAC from CHO cells into mouse A9 cells

To prepare mouse ES cells carrying MDR1-MAC, CHO cells carrying MDR1-MAC (CHO (MDR1-MAC, hCHR 7-. DELTA.MDR 1)1, 2, 4) obtained in [ D ] above were introduced into mouse A9 cells having a high minicell-forming ability with respect to mouse A9 cells by the micronucleus fusion method. A total of 7 resistant colonies obtained by 4 micronuclear cell fusions were separated and proliferated for subsequent analysis (clone name: A9(MDR 1-MAC)). As a result, 5 clones were positive in PCR using the above primers for detecting only the MDR1-MAC region. Furthermore, FISH analysis was performed using MDR1-BAC (RP11-784L5) (CHORI) and mouse minor satellite DNA probes (Tomizuka et al, Nature Genet.16: 133, 1997). As a result, the presence of MDR1-MAC specifically detected using the above probe was confirmed in 3 out of 5 clones. The following conclusions can be drawn as above: a9 cells carrying MDR1-MAC were obtained from 3 clones.

[F] Mouse ES cells carrying the mouse artificial chromosome vector MDR1-MAC were prepared as described in example 8, and the ES cells were used to examine stability in vitro. Furthermore, chimeric mice were prepared from the ES cells, and a mouse system TC (MDR1-MAC) in which MDR1-MAC was transmitted as progeny was prepared. In addition, the TC (MDR1-MAC) mouse system described above can be used to test the stability of MDR1-MAC in somatic cells. In addition, the TC (MDR1-MAC) mouse system can reproduce drug delivery in humans, and the like. Further, since the TC (CYP3A-MAC/MDR1-MAC) system can be prepared by mating with the TC (CYP3A-MAC) mouse system, it can be used as a model mouse for in vivo tests for investigating drug efficacy and toxicity in the development of pharmaceuticals.

Industrial applicability of the invention

The mouse artificial chromosome vector of the present invention has the same usefulness as the human artificial chromosome described in WO2009/063722, but can be stably carried in rodent cells and stably carry a target gene (group) for a long time by further increasing the carrying rate in rodent cells and rodent individuals, and can analyze the introduced gene between individuals and tissues more accurately because there is no variation in the amount of the introduced gene between individuals and tissues of rodents such as mice. The mouse artificial chromosome vector of the present invention can be used for various purposes and purposes, for example: introduction of a foreign gene into a recipient cell, utilization in establishment or regenerative medicine of iPS cells, production of a cell expressing a foreign gene and a useful non-human animal, production of a protein, analysis of gene function, and the like.

Deposit number

Deposit number of DT40B6 bT-1: FERMBP-11128

Sequence Listing free text (free text)

Sequence number 1 ~ 212: primer and method for producing the same

All publications, patents and patent applications cited in this specification are herein incorporated by reference as if fully set forth.

Claims (34)

1. A mouse artificial chromosome vector comprising a natural centromere derived from mouse chromosome 11, a long-arm fragment derived from mouse chromosome 11 obtained by deleting the distal end of the long arm from the long-arm part of mouse chromosome 11 in the vicinity of the centromere, and a telomere sequence, wherein the vector is stably carried in cells and tissues of a mammal having a carrying rate of 90% or more, and wherein the vector comprises, as a basic structure, the mouse artificial chromosome contained in deposited cell strain DT40B6bT-1 having the deposit number FERM BP-11128, wherein the mammal is a rodent selected from a mouse, a rat, and a hamster.

2. The mouse artificial chromosome vector of claim 1, further comprising one or more DNA sequence insertion sites.

3. The mouse artificial chromosome vector according to claim 2, wherein the DNA sequence insertion site is a recognition site of a site-specific recombinase.

4. The mouse artificial chromosome vector according to claim 2, wherein the DNA sequence insertion site is loxP sequence, FRT sequence, φ C31attB and φ C31attP sequence, R4attB and R4attP sequence, TP901-1attB and TP901-1attP sequence, or Bxb1attB and Bxb1attP sequence.

5. The mouse artificial chromosome vector of claim 1, further comprising a reporter gene, a selectable marker gene, or both.

6. The mouse artificial chromosome vector according to claim 1, further comprising an exogenous DNA sequence.

7. The mouse artificial chromosome vector according to claim 6, wherein the size of the foreign DNA sequence is 200kb or more.

8. The mouse artificial chromosome vector of claim 6, wherein the exogenous DNA sequence is a human DNA sequence.

9. The mouse artificial chromosome vector according to claim 6, wherein the foreign DNA sequence is a DNA sequence of a drug metabolism-related gene.

10. The mouse artificial chromosome vector according to claim 9, wherein the drug metabolism-related gene is a gene encoding an enzyme involved in the first phase reaction or the second phase reaction.

11. The mouse artificial chromosome vector of claim 10, wherein the enzyme involved in the first phase reaction is an enzyme encoding at least one selected from the group consisting of CYP1A, CYP1B, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2J, CYP3A, CYP4A, CYP4B and subfamilies thereof, and CES.

12. The mouse artificial chromosome vector according to claim 10, wherein the enzyme involved in the second phase reaction is an enzyme encoding at least one selected from the group consisting of UGT1 and UGT 2.

13. The mouse artificial chromosome vector according to claim 9, wherein the drug metabolism-related gene is a gene encoding a transporter.

14. The mouse artificial chromosome vector according to claim 13, wherein the gene encoding the transporter is a gene encoding at least one selected from the group consisting of MDR1, MDR2, MRP2, OAT, OATP, OCT, and BCRP.

15. The mouse artificial chromosome vector according to claim 9, wherein the drug metabolism-related gene is a gene encoding a nuclear receptor.

16. The mouse artificial chromosome vector according to claim 15, wherein the gene encoding the nuclear receptor is a gene encoding at least one selected from the group consisting of PXR, AhR, CAR, and PPAR α.

17. The mouse artificial chromosome vector according to claim 6, wherein the exogenous DNA sequence is a DNA sequence of a long arm or a short arm of a human chromosome.

18. The mouse artificial chromosome vector according to claim 6, wherein the foreign DNA sequence contains at least two gene sequences selected from the group consisting of a gene encoding an enzyme involved in a first phase reaction, a gene encoding an enzyme involved in a second phase reaction, a gene encoding a transporter, and a gene encoding a nuclear receptor.

19. The mouse artificial chromosome vector according to claim 17, wherein the DNA sequence of the long arm or the short arm of the human chromosome is a DNA sequence of the long arm or the short arm of the human chromosome containing a pathogenic region of a disease gene.

20. The mouse artificial chromosome vector according to claim 6, wherein the foreign DNA sequence is a sequence of a gene or DNA encoding a polypeptide selected from the group consisting of hormones, growth factors, nutritional factors, hematopoietic factors, coagulation factors, hemolytic factors, immunoglobulins, and G protein-coupled receptors, or a sequence of a gene or DNA for treatment associated with a disease selected from the group consisting of tumors, muscular dystrophy, hemophilias, neurodegenerative diseases, autoimmune diseases, allergic diseases, and other genetic diseases.

21. The mouse artificial chromosome vector according to claim 6, wherein the foreign DNA sequence is a sequence of a gene or DNA encoding a cytokine.

22. The mouse artificial chromosome vector of claim 6, wherein the foreign DNA sequence is a sequence of a gene or DNA encoding an enzyme.

23. The mouse artificial chromosome vector of claim 1, wherein the cell is a hepatocyte, an enterocyte, a renal cell, a splenocyte, a lung cell, a cardiac cell, a skeletal muscle cell, a brain cell, a bone marrow cell, a lymphocyte, a megakaryocyte, a sperm, or an ovum.

24. The mouse artificial chromosome vector according to claim 1, wherein the tissue is a tissue derived from a liver, an intestine, a kidney, a spleen, a lung, a heart, a skeletal muscle, a brain, a bone marrow, a testis, or an ovary.

25. A cell other than a germ cell, an Embryonic Stem (ES) cell, and an induced pluripotent stem (ips) cell carrying the mouse artificial chromosome vector of any one of claims 1-24, wherein the cell is of rodent origin, and the rodent is selected from a mouse, a rat, or a hamster.

26. The cell of claim 25, wherein the cell is selected from the group consisting of a somatic cell, a stem cell, and a precursor cell.

27. The cell according to any one of claims 25 to 26, wherein the cell is a primary culture cell, a secondary cell or a cell line.

28. The cell of claim 25, wherein the cell is a cell that produces a human antibody.

29. A method of producing a protein, the method comprising culturing the cell of any one of claims 25-28 carrying a mouse artificial chromosome vector comprising an exogenous DNA sequence, wherein the cell is of rodent origin and the rodent is selected from a mouse, rat or hamster, and recovering the protein encoded by the DNA produced.

30. A method for producing a human antibody, which comprises producing a human antibody using a non-human animal carrying the mouse artificial chromosome vector according to any one of claims 1to 24 containing a human antibody gene, wherein the non-human animal is selected from a mouse, a rat and a hamster, and recovering the antibody.

31. A method for testing pharmacological effects and/or toxicity of a drug or food, which comprises administering the drug or food in vitro to cells or tissues of a non-human animal carrying the mouse artificial chromosome vector according to any one of claims 1to 24 containing a gene related to human drug metabolism, wherein the cells or tissues of the non-human animal are derived from mouse, rat or hamster, and measuring the pharmacological effects and/or toxicity of the drug or food.

32. A method of testing drug or food toxicity, the method comprising: culturing a drug and/or food product and a cell or bacterial culture together with a non-human animal-derived microsome or microsome fraction S9, wherein the non-human animal-derived microsome or microsome fraction S9 is from a mouse, rat, or hamster and comprises the mouse artificial chromosome vector of any one of claims 1to 24 containing a human drug metabolism-related gene; and determining the effect of the drug or food product on the cell or bacterial culture.

33. A method for stabilizing large-sized DNA in a cell or an individual, the method comprising using the mouse artificial chromosome vector of any one of claims 1to 24, and stably maintaining large-sized foreign DNA of 200kb or more in a rodent cell or a rodent individual at a carrying rate of 90% or more, wherein the rodent is selected from a mouse, a rat, or a hamster.

34. A mouse artificial chromosome vector contained in the deposited cell line DT40B6bT-1 with the deposit number FERM BP-11128.