TWI223585B - Chimeric animal and method for producing the same - Google Patents

Abstract

A chimeric, non-human animal can be produced by a method that entails providing a microcell that contains one or more foreign chromosomes or fragment(s) thereof and then fusing the microcell with a pluripotent cell, thereby introducing the foreign chromosome(s) or fragment(s) into the latter. The pluripotent cell thus obtained can be used to generate a chimeric, non-human animal, the cells, tissues, and/or progeny of which can be the source of a product, such as an antibody, that is associated with one or more genes on the foreign chromosome(s) or fragment(s).

Description

1223585 A7 B7 V. Description of the invention (1) Technical scope The present invention relates to a chimeric non-human animal, its manufacturing method and its utilization method. If a non-human animal is combined after using the present invention, it has not been possible so far to make it possible to maintain and express a huge DNA fragment of 1 Mb (million test pairs) or more in an animal. Therefore, by using these, it is possible to produce an individual animal that expresses the full length of a gene that encodes a biologically active substance, such as a human antibody gene. Biologically active substances obtained from this animal, such as human-type antibodies, have utility value as pharmaceuticals. • It is possible to analyze the resistance of human huge genes (tissue suitability antigens, oxazine (^ 口 口 7, etc.) to individual animals.) • It can be used to make model animals of human priority genetic diseases and chromosomal abnormalities. Background Technology The technology that allows foreign genes to be expressed in individual animals, that is, the technology for making genetically modified animals, is not only used to obtain information about the in vivo functions of its genes, but also to identify the DNA sequences that control gene expression (for example, Magram et al., Nature, 3 15: 338, 1985), development of human disease model animals (Samura et al., Mammalian disease model mice, Zhongshan Bookstore, 1994), and breeding of livestock (eg, Muller et al., Experientia, 47: 923, 1991) is used for the production of useful substances (for example, Velander et al., PNAS ·, 89: 12003, 1992). As the target of gene introduction, mice have been used the most. As experimental animals, after detailed research, also The mouse with established embryo manipulation technology can be said to be the mammal most suitable for this purpose. -4- This paper size applies to China National Standard (CNS) A4 (210 X297 mm)

1223585 A7 B7 V. Description of the invention (2) The method of introducing foreign genes into individual mice can be divided into two types. One is a method of injecting DNA into a fertilization #pronucleus (Gordon et al., PNAS, 77:73 80, 1980), and the other is to introduce DNA into embryonic stem cells (hereinafter referred to as ES cells) that maintain differentiation and pluripotency. Chimeric mouse method (Takahashi et al., Development, 102: 259, 1988). In the latter case, in the chimeric mouse, only the donor cells and tissues of the ES cells, and the offspring derived from the germ cells derived from the ES cells, retain the introduced genes in all cells and tissues. Using these techniques, many transgenic mice have been produced to date. However, in the current situation, there is a limit to the size of DNA that can be introduced, which greatly limits the scope of use of this technology. The limit depends on the size of the cloned DNA. One of the largest DNA fragments introduced to date is the yeast. An approximately 670 kb DNA fragment cloned in the chromosome (YAC) (Jakobovits et al., Nature, 362: 255, 1993). This experiment was performed by fusing YAC-preserving yeast to mouse ES cells. In YAC, foreign DNA can be cloned to about 2Mb (Den Dunnen et al., Hum. Mol. Genet., 1:19, 1992). In budding yeast cells, it is known that the same DNA sequence has a high frequency of recombination with each other. It is difficult to keep human DNA fragments with many iterative sequences in perfect form. In fact, about 20 to 40% of the YAC gene bank containing human genomic DNA has undergone some recombination (Green et al., Genomics, 1: 1: 584, 1991). Another method is to microdissect the intermediate chromosomes obtained from human feeder cells and try to inject fragments (presumably 10Mb or more) into mouse fertilized eggs (Richa et al., Science, 245: 175, 1989). Mice obtained from this result -5- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (3) Individuals have detected human-specific DNA sequences (Alu sequences), and the human gene performance has not been confirmed. In addition, the chromosome preparation method used here, when the slide is fixed, the use of methanol acetate cannot avoid that the DNA itself becomes a small piece, and the possibility that the injected DNA exists as continuous DNA is low. In any case, so far, continuous foreign DNA fragments of more than 1Mb have been introduced into individual mice, and no case has been found. Useful and interesting human genes to be introduced into mice: antibodies (eg, Cook et al., Nature Genetics, 7: 162, 1994), T cell receptors (Hood et al., Cold Spring Harbor Symposia on Quantitative Biology, Vol. LVIII, 339, 1993), tissue-suitable antigens (Carrol et al., PNAS, 84: 853 5, 1987), and oxazine (Dennen et al., Preface), whose coding field is known to have a size of more than 1 Mb. In particular, since the importance of pharmaceuticals as human antibodies, it is desirable to make human immunoglobulin heavy chains (~ 1.5Mb, Cook, etc.), light chains / c (~ 3Mb, Zachau, Gene, 135: 167). , 1993), light chain lambda (~ 1.5Mb, Frippiat et al., Hun. Mol. Genet., 4: 983, 1995). And the performance of mice is impossible based on the current technology (Nikkei Biotechnology (A 彳 only in the evening), July 5, 1993). In addition, many of the genes that cause humans' priority genetic diseases and congenital abnormal chromosomal abnormalities (Down's disease, etc.) are not cloned, and can only be used for general location information on chromosomes. For example, G-bands obtained by staining metaphase chromosomes with gold (氺 厶 十) "generally have a size of Mb ~ 10Mb or more. Even if it is understood that a certain reason exists in a specific G-band, in order to For introduction of these abnormal shapes into mice, the cause gene-6 must be introduced-This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (4) Peripheral chromosome fragments (more than Mb), which is also infertile in current technology b. Here, we hope to develop a technology that introduces DNA beyond 1Mb, which is beyond the boundaries of the previous technology, into individual mice and expresses it. By culturing cells in animals, DNA of a size exceeding the above-mentioned limit can be introduced by conventional techniques. This process is performed using chromosomes (in the case of humans, with a size of about 50 ~ 3OOMb) as the medium. There are several reports on the method of chromosome transfer into cells (for example, McBride et al., PNAS, 70: 1258, 1973). Among them, as a method for introducing only one desired chromosome, the most suitable method is the microcellular method (Koi et al. Jpn. J. Cancer Res., 80: 413, 1989). A structure called a microcell is one in which several chromosomes are enveloped in the nuclear membrane and the plasma membrane. In a certain type of cell, micro-cells derived from an agent that inhibits the formation of pituitary gland are isolated and can be introduced into a few (in many cases, one) chromosomes by fusion with the recipient cell. The gene bank of single-chromosome hybrid cells with only one human chromosome obtained by this method can be used for the genetic mapping of known genes and the identification of chromosomes with unknown cancer suppressor genes and cell aging genes (for example, Saxon Et al., EMBO J., 5: 3461, 1986). In addition, by irradiating r-rays to microcells, chromosomes can be fragmented and introduced into a part of them. (Koi et al. Science, 260: 361, 1993). That is, the microcell method can be considered as a method suitable for introducing DNA of 1 Mb or more into animal culture cells. Whether self-cultured cells can make the If expectation in mice and stably maintain the performance of differentiated totipotent ES cells (Evans et al., Nature, 292: 154, 1981) is indeed a reality. Here ES cells can be introduced from outside. The paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) binding.

Line 1223585 A7 B7 V. Description of the invention (5) Sudden mutations in genes and species, moreover, mutations in the target gene recombination, genetic changes at the individual level of the mouse, can be performed freely (eg, Mansour et al., Nature, 336: 348, 1988). On the one hand, the significance of the introduction of huge DNA can be considered as the size of the optional foreign DNA fragments in the YAC vector described above. The technique of transferring DNA exceeding its size into the chromosome of cultured cells has not been used. In the introduction of genes at the individual level of mice, and its realization is also considered difficult (Muramatsu et al., Transfer of gene biology (Bu Jin y only ^ Gong Er 7 Xi A 彳 口 一 1), talk about social science (inch彳 y (7) (P143-, 1989). The following can be cited as reasons. • When the recipient cell is a mouse ES cell with a normal karyotype and a human chromosome is introduced into it, it is a person with an abnormal chromosome introduction. So far, the most significant genetic abnormalities of chromosomes that can be identified by a microscope have been fatal in many cases in mice (Gropp et al., J. Exp. Zool ·, 228: 253, 1983; Xiang Yi Shen Yi , Biomanipulation Series (〆 彳 才 ^ 二 二 了 瓜 シ W — Again ') 8 gene target, Yangtusha, 1995). • The human chromosomes available to us are usually normal fibroblasts with limited proliferation, or differentiated somatic cells such as cancer cells. When chromosomes from such somatic cells are introduced into undifferentiated ES cells, consideration can be given to whether ES cell differentiation (Muller et al., Nature, 3 1 1: 438, 1984), aging (Sugawara, Science, 247: 707, 1990) 〇 The case where chromosomes from somatic cells are introduced into the early stage embryos of tobacco, for the embryogenesis process Whether it can function normally as the originator of germ cells, and can be used for specific gene expression in a variety of tissues and cells. -8- This paper applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1223585 A7 B7 V. Description of Invention (6) Very little research. One of the big differences between these two can be imagined as the methylation status of chromosomal DNA. This change accompanying cell differentiation suggests an important role in tissue-specific gene expression (Ceder, Cell, 53: 3, 1988). For example, for the site-specific DNA recombination reaction that is indispensable for the activation of antibody genes, when a methylation matrix is introduced into B cells, the state of methylation is also maintained after replication, and reports of inhibition of the recombination reaction (Hsieh et al. , EMBO J., 11: 315, 1992). Furthermore, de novo methylation also occurs in strained cells compared to in vivo (Antequera et al., Cell, 62: 503, 1990). From the studies so far, it is not easy to imagine that the fibroblasts and human mouse hybrid cells may be highly methylated antibody genes that are normally expressed in mouse B cells. Here, the two reports of Illmensee et al. (P.N.A.S., 75: 1914, 1978; P.N.A.S., 76: 879, 1979) cannot be ignored. One report was made from human tumor cells and mouse ES cells, and the other was made from chimeric mice produced by fusion of rat liver cancer cells and mouse EC cells. In these reports, the experimental results have been criticized for many doubts and considered to be of low reliability (Noguchi et al., Teratoma of mice, Science and Engineering Society, Section 5, 1987). In addition, although it is hoped that the test can be replenished as soon as possible, there have been no reports of this experiment since 17 years to the present. Therefore, with these reports, it is not possible to maintain foreign chromosomes in individual mice and to express genes on the chromosomes. In this situation, it has been difficult to introduce the chromosomal fragment Chengpi's huge DNA into individual animals such as mice. In fact, there has been no review of this issue since the report by Illmensee et al. -9-This paper size applies to Chinese National Standard (CNS) A4 (210X297 mm) 1223585 A7

Therefore, the present invention aims to provide a method for producing a chimeric non-human animal and its offspring that retains a foreign chromosome or a fragment thereof, a gene expressed on the dye or the fragment thereof, and a magical non-human animal. The present invention also aims to provide tissues and cells derived from the chimeric non-human animal and its offspring. It is another object of the present invention to provide a hybrid tumor produced by fusion of a chimeric non-human animal and its offspring podocytes and myeloid tumor cells derived from the foregoing. In addition, the present invention aims to provide a method for producing a biologically active substance using an individual, tissue or cell of a non-human animal as a product of expression of a gene on a foreign chromosome or a fragment thereof. DISCLOSURE OF THE INVENTION In order to solve the above-mentioned problem, the inventor deliberately researched the result, and introduced the chromosome from human normal fibroblast cells or a part of the fragment into mouse ES cells by a microcell method, and successfully obtained a strain that stably maintained . In addition, from this ES cell line, human chromosomes are retained in normal tissues, and chimeric stylized expression of human genes containing a plurality of human antibody heavy chain genes is obtained. With this series of methods, we have made it impossible for individuals to retain huge DNA fragments and express genes on the DNA fragments. The gist of the present invention is as follows. (1) It is characterized by the production of microcells containing single or plural foreign chromosomes or fragments, and by fusion with the microcells, the former single or plural foreign chromosomes or fragments are transferred to cells with multipotential potential. Non-human animal production method. -10- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

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Line 1223585 V. Description of the invention (2) "To make microcells containing single or plural foreign chromosomes or fragments thereof, the fusion of the fine cells with the microcells, so that the preceding single or plural non-stains are stained, or the fragments are moved to retain multiple points. It is characterized by the cells with potential-changing potential, and the method of making cells with multiple differentiation potentials containing early one or plural foreign chromosomes or fragments thereof. (3) The method described in (1) above can be used to make and retain single or plural foreign chromosomes. Or a fragment thereof, and then expressing the gene on the chromosome or a fragment thereof to a non-human jaw animal, and retaining the single or plural exotic chromosome or a fragment thereof described above, and expressing the gene on the child's chromosome or a fragment to its offspring. 4) The method of the preface (2) can be used to make and maintain the cells with multiple differentiation potentials containing single or plural exotic chromosomes or fragments thereof. (5) The multi-differentiation of the preface (4) can be kept for making chimeric non-human animals. Use of potential cells. (6) A single or plural foreign chromosome or fragment obtained from the mating of a chimeric non-human animal or its offspring in (3) above. Non-human animals and their offspring represented by genes on chromosomes or fragments thereof. (7) Non-human animals or their offspring from other brothers (3) or the tissues of non-human animals or their offspring from the previous (6). 8) Cells from chimeric non-human animals or their offspring from the previous paragraph (3) or non-human animals or their offspring from the previous paragraph (6). (9) Made by fusion of the cells of the former (8) and myeloma Hybridomas. One (10) The chimera non-human animal or its descendants of the preface (3) or the non-human animal or its descendants of the preface (6), so as to make the beauty on the chromosome or fragment of the preface -11-^ paper Standards applicable to SS home fresh specifications (21GX 297 male ^ '-------- 1223585 A7 B7 V. Description of the invention (9

Mating of non-human animals caused by the same or similar genetic defect system, non-human animals or their offspring. (11) A chimeric non-human animal or its offspring or the individual, tissue, or cell of the non-human animal or its offspring of (6) above, using a single or plural gene on a chromosome or a fragment thereof The method of manufacturing biologically active substances characterized by biologically active substances recovered as their products. (12) A system in which the chimeric non-human animal or descendant of the preceding paragraph (3) or the non-human animal or descendant of the preceding paragraph (6) is deleted from the same or similar gene as the gene on the chromosome or fragment thereof. The mating of non-human animals, the individual animals, tissues or cells of the raw animals can be used to stain the genetic expression of Xiaodou or its fragments in the single or plural of the previous description, and to recover the biological and active substances of its products. Manufacturing method of biologically active substances. Brief Description of the Drawings Fig. 1 shows the results of analysis (PCR analysis) of 89 cells with human second chromosome (fragment). Figure 2 shows that the human chromosome 22 (fragment) is retained in the E14-resistant strain (PCR analysis). Fig. 3 is an electrophoretic photograph showing the human L1 sequence in a chimeric mouse derived from ES cells introduced into chromosome 22 (analysis by southern aspiration method). Figure 4 is an electrophoretic photograph (PCR analysis) showing tf in a human mouse chromosome-introduced chimeric mouse organ containing human chromosomes. (Panel) is an electrophoretic photograph showing the results of human gene expression (RT-PCR) of tf in chimeric mice introduced into human chromosome 22. -12- This paper size applies to China National Standard (CNS) A4 (210X 297 public love)

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Line 1223585 A7 B7 V. Description of the invention (10) Figure 6 is an electrophoretic photograph showing the results of human gene expression (RT-PCR) in a chimeric mouse organ introduced into human chromosome 22. Figure 7 shows that the human chromosome 4 (fragment) is retained in the E14-resistant strain. FIG. 8 is an electrophoretic photograph showing the results of the detection of the human L1 sequence (analyzed by the southern aspiration method) in the E14 cell line introduced into the human chromosome 4. Fig. 9 is an electrophoretic photograph showing the human L1 sequence in a chimeric mouse derived from ES cells introduced into the human chromosome 4 (analysis by southern aspiration method). Fig. 10 shows that the human chromosome 14 (segment) is maintained in a TT2-resistant strain (PCR analysis). Fig. 11 is an electrophoresis photograph (PCR analysis) showing a human chromosome-like pattern after being introduced into human chromosome 14 from es cells. Fig. 12 is a graph showing the results of the resistance test of fibroblasts G41 8 from the tail. Figure 13 shows the human antibody IgM concentration (ELISA) in the serum of human serum albumin-immunized chimeric mice. Fig. 14 shows the concentration of human antibody IgG (ELISA) in the serum of human serum albumin-immunized chimeric mice. FIG. 15 shows the results of the analysis (ELISA) of H4B7, a hybridoma produced by human IgM. Fig. 16 is a photograph showing the chromosome morphology of the results obtained by FISH analysis of a mouse ES cell line (TT2 cell line pQi5) containing a partial fragment of human chromosome 2 and a partial fragment of chromosome 14. Figure 17 shows the antiserum in the serum of human serum albumin-immunized chimeric mice. -13- This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 $ $ --- ---)

Line 1223585 A7 B7 V. Description of the invention (11) Increase in human serum albumin and human IgG antibody. Fig. 18 is a graph showing an increase in the value of anti-human serum albumin human Ig / c antibodies in the serum of human serum albumin-immunized chimeric mice. Fig. 19 is an electrophoretic photograph showing the results of detection of human L1 sequences (analyzed by southern aspiration method) in a TT2 cell line introduced into human chromosome 22. Figure 20 is a graph showing an increase in the value of anti-human serum albumin human Ig λ antibody in the serum of human serum albumin-immunized chimeric mice. Fig. 21 shows the offspring of a chimeric mouse introduced with a partial fragment of the human chromosome 2 and retaining a partial fragment of the human chromosome 2 (PCR analysis). FIG. 22 shows the presence of human #chain cells on the surface of the spleen of a chimeric mouse introduced into human chromosome 14 (analysis of a flow cell assay (7 mouths of an inch) / b). FIG. 23 Fig. 24 shows the structure of LoxP-pstNEO plastid DNA. Fig. 24 shows the structure of genomic DNA of mouse antibody heavy chain C #. Fig. 25 shows the structure of genomic DNA of mouse antibody light chain C / c. 26 In order to show the vector with mouse antibody heavy chain labeling and the DNA fragment that can be detected by the recombinant with the same structure as the probe used in the southern suction method. Figure 27 shows the vector with mouse antibody light chain / c labeling. DNA fragments that can be detected by recombinants with the same structure as the probes used in the Southern suction method. Figure 28 shows the southern parts of mice with the same heavy chain-resistant recombinants and high-concentration G41 8-tolerant strains from the same recombinants. An electrophoretic photograph of the results of the aspiration analysis.. _ The best form for carrying out the present invention is to retain the human chromosome or its fragment, and the base on this chromosome or its fragment is -14- This paper applies Chinese National Standard (CNS) A4 Specifications (210 X 297 mm) 1223 585 A7 B7 V. Description of the invention (12) Individual mice can be produced by (1) chromosome donor cells with labeled human chromosomes or fragments thereof (2) human chromosomes or The fragments were transferred into mouse cells with multipotential potential (3) Chimera mice using the mouse cells described above (4) Obtained through the process of human chromosome retention and human gene expression confirmation of the chimeric mice. Here, as a non-human animal that retains a human chromosome or a fragment thereof, and expresses genes on the chromosome or a fragment thereof, a mouse is taken as an example (hereinafter, this mouse is referred to as a "introduced human chromosome mouse"). Here, the human staining system refers to a complex of natural DNA and proteins from human cells. There are 23 kinds of normal human chromosomes (24 kinds of males) and 46 pieces of DNA, each containing a length of about 50 ~ 300Mb. As an independent chromosome in the present invention, it also contains some fragments that can be stably reproduced and distributed, and transferred to mice. A fragment of a chromosome. The size of the DNA is usually more than 1 Mb, and it may be smaller than this. The features of the present invention are not treated with colonization of coliforms, yeasts, etc. or extraction of DNA from cells, and the chromosomes themselves are used as mediators to allow human genes to be retained and expressed in mouse individuals. In addition, a mouse introduced into a human chromosome refers to a single or plural human chromosome or a fragment thereof retained in all or part of normal somatic cells. In addition, it refers to all or part of its normal somatic cells, expressing single or plural human genes on the human chromosome. (1) Reserve the chromosome of the labeled human chromosome or its fragments. -15- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1223585 A7 ___ B7 V. Description of the invention (13) To make donor cells for chromosomes, 1) retain human chromosomes labeled with markers that can be selected by recipient cells, 2) do not contain human chromosomes other than 3, and 3) microcells can form high-energy cells. As materials for providing human chromosomes, all human cell lines, cancer cells, and primary cultured cells can be used. However, considering that the possibility of abnormalities such as deletion and expansion of chromosomes is low and easy to cultivate, normal fibroblast cells are used as suitable. First, about 1) human cells can be expressed by drugs (G4 18, puramycin ((£ 21) v < eve >), hygromycin (/, 彳 义 '口 7 彳 夕) > ), Germ cytokine (Y today only 4 ::; >)) tolerance and other marker gene transformations. It is used here as a control marker. The promoter used for expression is not only human cells, such as mice Recipient cells of ES cells are expected to be highly efficient. For this purpose, a linker of SV40-enhancing factor and the simple herpes virus thorakinase promoter gene can be used (Katoh et al., Cell. Struct. Funct., 12: 575 , 1987), the stingy PGK-1 promoter gene (Soriano et al., Cell, 64: 693, 1991), etc. The shape conversion was performed by the electroporation method (Ishida et al., Introduction to Cellular Engineering Experiment Operation, Kodansha '19 9 2), etc. By choice, the introduced marker genes can be used to generate gene libraries of human cell transformants randomly inserted on 23 kinds of 46 human chromosomes. About 3) Many human normal cells have low microcell productivity, so A cell that forms a southern cell by transforming the upper body of the transformant and the microcell, such as Xiaoqi A 9 Whole cell fusion of cells (Oshimura, M., Environ, Health Perspect., 93:57, 1991) can impart formation energy. In mouse-human hybrid cells, -16- This paper size applies to China National Standard (CNS) A4 (210X297 mm)

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Line 1223585 A7 B7 V. Description of the invention (14) Knowing that the human chromosome has selectively disappeared, the fusion strain obtained here can be selected with the above-mentioned markers, which can stably retain the genetically mapped human chromosome. In addition, in order to satisfy the condition of 2), it is expected that microcells are obtained from this cell fusion strain and re-fused with mouse A9 cells. In this case, too, the microcell fusion strains obtained by selecting the markers on human chromosomes are those that satisfy the conditions of 1), 2), and 3). Identification of genetically mapped human chromosomes, and mouse-human single-chromosome hybrid cells obtained at the final stage can be analyzed by PCR (Polymerase Chain Reaction, Saiki et al., Science, 239: 487, 1988), Southern blot method (Ausubel et al., Current Experimental Projects in Molecular Biology, John Wiley & Sons, Inc., 1994), FISH (Fluorescence Optical Field Hybridization, Lawrence et al., Cell, 52:51, 1988) and other investigations. It is hoped that for the introduction of a specific chromosome, for many human cell transgenic asexual reproductive lines, the process described above will be repeated, and the target chromosome will detect the genetically located person. Alternatively, a mixture of human cell transformant asexually propagating lines can be subjected to the procedure described above, and the majority of the obtained mouse-human single-chromosome hybrid cells can be identified for human chromosomes. In addition, by the same recombination with the DNA sequence on the chromosome to be introduced as the target (Thomas et al., Cell, 5: 503, 1987), a marker gene can also be inserted at a specific position. Micro-cells prepared from mouse-human hybrid cells are irradiated with T-rays, and the genetically mapped human chromosomes are fragmented for introduction into mouse A9 cells. In addition, there is no case where micro-cells are irradiated with r-rays, and a portion of the fragmented human chromosome is transferred to a certain ratio. In these cases, the resulting microcell fusion strain retains a portion of the genetically mapped human chromosome. These can be used in the case where it is intended to apply the Chinese paper standard (CNS) A4 (210X 297 mm) 1223585 A7 B7 to this paper size. 5. Description of the invention (15) Part of the chromosome is introduced into the recipient cell. (2) The migration of human chromosomes or fragments thereof into mouse cells that retain multipotentiality has so far been reported by using mouse embryonic cancer cells (EC cells, Hanaoka et al., Differentiation, 48:83, 1991 from various systems). ), Embryonic stem cells (ES cells, Evans et al., Nature, 292: 154, 1981), embryonic germ cells (EG cells, Matsui et al., Cell, 70: 841, 1992) are injected into or co-cultured with early embryos. Helping with normal somatic cells, that is, making chimeric mice. ES and EG cells have particularly high abilities and can also help germ cells in many situations. They can produce offspring from cells. EC cells are mainly derived from germ cell carcinoma, ES cells are derived from the inner cell mass of placental cells, and EG cells are derived from the primitive germ cells that appeared in the early stages of development. As the recipient cells to which the human chromosome is transferred according to the present invention, these cell lines and variants thereof can be used, and all undifferentiated cells that can be differentiated into all or part of normal somatic cells in a mouse individual. As the material of the recipient cell into which the human chromosome is transferred, microcells prepared from the human chromosome donor cell obtained in (1) or microcells irradiated with 7 rays can be used. Recipient cells were transferred by fusion of the recipient cells with microcells according to the method described in < Shimizu Line, Handbook of Cell Engineering, Yangtusha, 1992 > and the like. For microcell donor cells, a certain human chromosome or fragment thereof is retained in the recipient cell with optional markers. From these, all human chromosomes, or fragments thereof, can be introduced by selecting a strain that retains the gene or chromosome or fragment thereof for the purpose of introduction by PCR, Southern blot analysis, FISH analysis, etc. as in (1). In addition, by sequentially introducing -18 to retain different selection marks, this paper size applies the Chinese National Standard (CNS) A4 (210 X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (16) A plurality of chromosomes or fragments thereof can also retain these recipient cells. In addition, among the cell lines that have been introduced into a certain human chromosome, the number of introduced chromosomes may be increased. This is usually achieved by increasing the concentration of the selected agent added to the culture medium. Recipient cells selected by markers on the human chromosome (G418 tolerance, etc.) retain all or part of the human chromosome retained by the donor cell, as confirmed below. Using genomic DNA extracted from selected recipient cells as probes, using human specific repeat sequences (L1, Alu et al., Korenberg et al., Cell, 53: 391, 1988), human genes, etc. Law analysis and detection. In addition, chromosomal analysis using fluorescence gene hybridization (FISH), etc. using human gene-specific primers and fluorescence spot hybridization (FISH) using human chromosome-specific probes (Lichter et al., Human Genetics, 80: 224, 1988) can be confirmed. . (3) Chimeric mice were prepared from ES cells introduced into human chromosomes. Chimeric mice were prepared from ES cell strains obtained in (2), and <Asagi Shinichi, Biological Manipulation Series 8 Gene Target, Yangtusha, 1995 &gt; And so on. For the selection of the generation period, system, and the like of host embryos for efficient chimeric mouse production, it is desirable to use the conditions reviewed for the ES cell line, respectively. For example, for TT2 cells from CBAX C57BL / 6 F1 (wild color, Yagi et al., Analytical Biochemistry, 214: 70, 1993), use Balb / c (white, Japanese Krya) or ICR (white, Japanese Krya) Company) of eight cell_phase embryos is expected as a host embryo. (4) Conservation of human chromosomes in chimeric mice and expression of human genes in ES cells derived from embryos injected into ES cell lines, -19- This paper applies Chinese National Standard (CNS) A4 Specifications (210 X 297 mm) Staple

Line 1223585 A7 B7 5. Description of the invention (17) It can be roughly judged from its coat color. However, you cannot judge that you have no contribution to coat color, that is, you have no contribution to other organizations. More specifically, the retention of human chromosomes in various tissues of the chimeric mouse can be confirmed by Southern blot analysis and PCR using genomic DNA extracted from each tissue. The expression of genes introduced into human chromosomes can be confirmed as follows. The expression of mRNA from human chromosomes was detected by RT-PCR method using RNA from various tissues (Kawasaki et al., P.N.A.S., 85: 5698, 1988), Northern suction method (Ausubel et al., Supra). The expression of protein level is based on the enzyme immunoassay of anti-human protein antibody (ELISA, Toyama • Anton, monoclonal reproduction antibody antibody experimental operation, which minimizes the cross-reactivity of the same protein with mice, talk about social sciences, 1987; Ishikawa, Ultra-High Sensitivity Enzyme Immunoassay, Society Publishing Center, 1993), Western Aspiration Method (Ausubel et al., Foreword), or Isozyme Analysis Using Different Electrophoresis Degrees (Koi et al., Jpn · J · Cancer Res ·, 80: 413, 1989) and so on. In addition, the retention of the human chromosome in the chimeric mouse cell and the expression of the gene on the chromosome can be confirmed by the appearance of the resistant cell represented by the drug resistance marker gene from the primary cultured cell of the chimeric mouse. For example, for chimeric mice made from ES cells that retain human chromosome 14 in which human immunoglobulin heavy chains are present, human IgM, IgG, IgA, etc. in the serum of the chimeric mouse can be combined with mouse antibodies. Enzyme immunoassay with minimal cross-reactivity to anti-human Ig antibodies. In addition, this chimeric mouse is immunized with human-derived antigens (such as human_serum albumin), and hybrid tumors (Anton, Chiba, and monoclonal antibody produced by fusion of its spleen cells with mouse myeloma) Introduction to experimental operation, talk about social science, -20- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (18) 1991) By ELISA screening, hybridomas producing human immunoglobulin heavy chains can be obtained. In the above, a mouse is taken as an example to explain a method for preparing a chimeric human animal that retains a human chromosome or a fragment thereof, and expresses genes on the chromosome or a fragment thereof. In the present invention, a chromosome or a fragment of a chimeric non-human animal is transferred. It is not limited to those from human beings. A wide range of foreign chromosomes or fragments can be transferred to make the genes on the chromosomes or fragments appear. Here, the "foreign chromosome" is characterized by the expression of genes on the chromosome or a fragment thereof in a chimeric non-human animal by being transferred into a cell that retains the potential for multi-differentiation, and there is no particular limitation on its origin. In addition, according to the method of the present invention, not only chimeric mice but also other chimeric animals, such as mammals of rats, threads, etc., can be produced. The establishment of ES cells or ES-like cells in animal species other than mice is reported in rats (Iannaccone et al., Dev. Biol., 163, 288-, 1994), pigs (Wheeler et al., Reprod, Fertil · Dev ·, 6,563-, 1994), cattle (Sims et al., Proc. Natl. Acad. Sci. USA, 91, 6143-6147, 1994), and also tried fish, difficult, etc. (transgenic animals, protein nucleic acid enzymes, 1995 Supplement to the October issue, published by Kyoritsu). In addition, it is known that transplantation of unfertilized eggs from the nucleus of epithelioid cells obtained from ES-like cells (ED cells) and subsequent generations of more than 10 generations occurs normally (Campbell et al., Nature, 3 80, 64 -, 1996). By transferring these ES or ES-like cells as recipient cells, foreign chromosomes can be transferred. As in the case of mice, the foreign chromosomes and fragments thereof can be retained, and chimeric non-expressions of genes on the chromosomes or fragments can be made. Human animal. Moreover, in the present invention, the multi-differentiation of foreign chromosomes or fragments thereof transferred is retained-21-This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1223585 A7 B7 V. Description of the potential of the invention (19) The cells are not limited to the aforementioned ES cells, EC cells, and EG cells. For example, a foreign chromosome or a fragment thereof can be transferred into a bone marrow stem cell, and a living body implanted in the bone marrow stem cell can be used to treat a genetic disease or the like. In the case of chimeric non-human animals, ES cells that retain foreign chromosomes differentiate into their germ cells. Offspring derived from reproduction are also confirmed to be introduced into chromosomes or fragments, and their offspring will be genetically expressed on the introduced chromosomes or fragments. By using the chimeric non-human animal or its offspring obtained as described above, the genes on the foreign chromosome or its fragments can be expressed, and the biologically active substance can be manufactured by recovering the product. Specifically, an individual of a chimeric non-human animal or its offspring is bred under conditions such that genes on foreign chromosomes or fragments thereof can be expressed, and thereafter expression products can be recovered from blood and ascites of animals. Or chimeric non-human animals or their offspring's tissues, cells, or immortalized them (for example, hybridomas that are immortalized by fusion with myeloma) are equal to those that can express genes on foreign chromosomes or fragments thereof After culturing under conditions, performance products can be recovered from the culture thereafter. Furthermore, since these chimeric non-human animals or their offspring's tissues, cells, or DNA composed of foreign chromosomes or fragments extracted from them, will remain in the chimeric non-human animals or their offspring's tissues , Cells, or cDNAs derived from foreign chromosomes or fragments thereof from the immortalized ones into animal cells or insect cells (for example, CHO cells, BHK cells, liver cancer cells, myeloma cells, SF9 cells, etc.), Cells are cultured under conditions in which genes on foreign chromosomes or fragments thereof can be expressed, and thereafter, expression products (such as specific antigen-specific antibody proteins, etc.) can be recovered from the culture. The performance products can be recovered by well-known methods such as centrifugal separation. In addition, they can be separated and divided according to ammonium sulfate differential. 22- This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1223585 A7 B7 V. Invention Explanation (20) Refined by well-known methods such as compounding chromatography, gel filtration chromatography, adsorption chromatography, and thin-layer chromatography. Biologically active substances include all substances encoded on foreign chromosomes, such as antibodies, especially human antibodies. For example, spleen cells of chimeric animals or their hybridomas can be self-immortalized, and human antibody genes on the chromosome can be cloned and introduced into Chinese hamster ovary cells (CHO) and bone marrow cell tumors to produce human antibodies (Lynette Et al., Biotechnology, 105 1 12 1-, 1992; Bebbington et al., Biotechnology, 10, 169-, 1992). Chimeric mice and their offspring that retain human chromosomes 2, 14, and 22 obtained according to the present invention can retain human antibody heavy chains (on chromosome 14) and light chains / C (on chromosome 2). ), Light chain; L (on chromosome 22) most of the functional sequences of the respective genes. That is, compared with known transgenic mice (Green et al., Nature Genetics, 7, 13-, 1994, Lonberg et al., Nature, 368, 856-, 1994, etc.) that use yeast artificial chromosomes to introduce a part of human antibody genes, Closer to the human observer, it can show a very diverse range of human antibody performances. Also, at the same time, the chimeric mice and their offspring of the chromosomes (fragments) of the 2 + th, 14th, 22 + 14th, etc. obtained according to the present invention are retained, and the 2 + th, 14th, obtained by mating them are retained at the same time. + Mice and offspring of chromosomes (fragments) of the 22nd order, both heavy and light chains can produce complete human antibodies from humans. These mice treat antigens from humans as foreign bodies to elicit an immune response and produce antigen-specific human antibodies. This property is very useful for the treatment of human single-source antibodies or human multi-source antibodies (Green et al., Former, Lonberg et al., Former). On the one hand, for specific antigens in order to improve the affinity. -23- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm)

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Line 1223585 A7 B7 V. Description of the invention (21) The efficiency of human antibodies is expected as a mouse that does not produce mouse antibodies and only produces human antibodies (Green et al., Former, Lonberg et al., Former). In the present invention, it is achieved by the following method A or B using a typical known method. Method A: A method using mouse antibody-deficient ES cells and a mouse antibody-deficient chimeric host embryo. Method B: A method of mating with a mouse system lacking a mouse antibody gene can be obtained by using a chimeric mouse introduced into a human chromosome to retain offspring of human chromosomes. The typical examples of the methods of A and B are described below. Specific Procedure of Method A 1. One of the mouse antibody heavy chain genes of two replicas present in mouse ES cells was disrupted using the target gene-like recombination method (Joyner et al., Gene Targeting, 1993, IRL PRESS). The gene is destroyed by site-specific recombination, and sandwiched between removable sequences such as ΙΟχΡ sequence (recombined by Cre recombinase, Sauer et al., Preface, others use FLP recombinase FRT sequence O'Gorman et al., Science, 251 (Example of 1351, 1991) Insertion of marker genes such as G4 18 resistance gene. 2. One of the antibody heavy chain genes was destroyed. The ES cell of the drug resistant mouse was cultured in the presence of a high concentration of the drug, and a strain resistant to the high concentration of the drug was selected. These strains can be screened to obtain strains in which the antibody heavy chain genes of both parties have been destroyed (Shinichi Shinichi, former note). 3. In mouse ES cells in which both of the antibody heavy chain 'genes of the two parties were destroyed, an enzyme gene that undergoes a site-specific recombination reaction between the recombination sequences inserted on both sides of the drug resistance gene of 1, For example, Cre recombinase gene-24- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) m binding

Line 1223585 A7 B7 V. Description of the invention (22) (Sauer et al., Foreword), temporarily introduced, a recombination reaction occurs between ΙχχΡ sequences, and a drug-susceptible strain that removes the drug resistance gene inserted in the antibody heavy chain genes of both parties is selected (Gaojin Shengzhi et al., Separate volume of experimental medicine, basic technology of immunological research, ρ255-, 1995, Yangtusha). 4. For the mouse antibody light chain / c gene, repeat the process of 1 to 3 described above to obtain the drug-sensitive strain that completely lost the antibody heavy chain and / c chain. 5. By fused with microcells using strain 4 (antibody heavy chain, / c chain-deleted mouse ES cells) as the recipient cells, and using drug resistance markers (such as the G418 resistance gene), humans containing genetically targeted cells will be included. Introduction of human heavy chromosome 14 (fragment) of antibody heavy chain gene. 6. Through microcell fusion using the strain obtained in 5 as the recipient cell, a gene containing a resistance marker different from 5 (for example, ptomycin (dose 2 v4shiy)) is used for gene mapping. The human chromosome 2 (fragment) or chromosome 22 (fragment) or both of the human antibody light chain gene is introduced. 7. Defective mouse systems such as RAG-2 (/ 7 eve of the 々B) Mice, Shinkali et al., Cell, 68, 855-, 1992, Membrane // Chain Defective Mice, Kitamura et al., Nature, 350, 423-, 1991) The embryos obtained were used as host embryos, and 6 and 6 were obtained. Chimeric mice of ES cells. 8. In the obtained chimeric mice, most of the functional B lymphocytes were derived from ES cells (Takajin Seki et al., Sep. of Experimental Medicine, Basic Technology of Immunology Research, p234-, Yangtusha, 1995). Here, due to the absence of the mouse heavy chain and / C chain, the lymphocytes of the B lymphocyte are mainly derived from functional human antibody genes introduced into the chromosome, producing only human antibodies. Procedures for Method B -25- This paper size applies to China National Standard (CNS) A4 (210X 297mm) binding

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1 · Self-preserving human staining with human technology to remove μ, body or its fragments, heavy chain, light chain κ or light chain λ to Sun Erqi Caution, you can keep them safely and replace them with Jiang Er : 1st place, the mouse system of human antibody heavy or light chain expression, or the human antibody heavy chain obtained, or the light chain, both of which represent a lineage, which is sufficient for the lack of their own antibody gene. Mating with Tibetan systems (such as membrane-type # chain-deficient mice, preface, _ Hammer ^ Han Gongquan "Xiara Xiaobao, Chen et al., EMB0 ,, 82_1, 1993), the mouse antibody heavy chain, and light The deletion of chain κ is a homozygous conjugation, and it contains human antibody heavy chain (14th) + light chain / c (second): antibody heavy chain (14th) + light chain λ (22) or human antibody heavy The mouse system of human chromosomes with chain (14th) + light chain / c (2) + light chain λ (22). In this mouse system, only the human antibody is produced due to the deletion of the mouse antibody heavy chain and light chain kappa genes' mainly from functional human antibody genes introduced into the chromosome. Furthermore, the methods A and B described above can be used because not only human antibodies are obtained, but also all gene products existing on foreign chromosomes efficiently. The following shows specific examples, but the present invention is not limited to these examples. (Example 1) The chromosome of human chromosome (fragment) labeled with G41 8 resistance was reserved for the production of cells to restrict the enzyme Sail (Takara Shuzo). The plastid pSTneoB containing the G418 resistance gene (Katoh et al., Cell Struct, Funct ·, 12: 575, 1987; Japanese Collection of Research Biologicals (JCRB), Deposit Number: VE039) linearized and introduced into human normal fiber bud cells HFL- • 26- This paper is in accordance with China National Standard (CNS) A4 (210X297) Public love)

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1223585 A7 B7 V. Description of the invention (24) 1 (purchased by RIKEN Cell Bank, RCB0251). HFL-1 cells were treated with trypsin, suspended in Dalbeco (Eve \ 7: ^) phosphate buffered saline (PBS) to 5xl06 cells / ml, and then a gene pulser (Bayou) was used in the presence of 10 μg of DNA. Lei (Hachiman Saijin v Bu ')), performed the electroporation method (Ishida et al., Introduction to Cellular Experiment Operation, Kodansha, 1992). The following 25 // F capacity is applied by using a 4 mm long electroporation cell (165-2088, Biole) at a voltage of 1000V and room temperature. Electroporation-treated cells were seeded in 3 to 6 100 mm tissue culture plastic culture dishes (15% bovine fetal serum (FBS) -containing Iggler-Small) F12 medium (hereinafter referred to as F12). Kolin (mouth one two '//)). In the future, it was replaced with F12 medium containing 15% FBS containing G418 (GENETICIN, Xi Goma 0: / Xi · '^) containing 200 µg / ml. After 2 to 3 weeks, 100 groups of the generated communities were taken as a group, and they were gathered into 52 groups, and then sown in 100 mm culture orphans and cultivated. Mouse A9 cells (Oshimura, Environ, Health Perspect., 93:57, 1991, JCRB0211) were added to Dalbeco-modified Iggler medium (hereinafter referred to as DMEM) supplemented with 10% bovine fetal serum (FBS), Cultivate in a 100 mm culture orphan. 52 groups of G418-resistant HFL-1 cells were cultured in 100% millimeters of F12 supplemented with 15% bovine fetal serum (FBS) and 200 micrograms / ml of G418. After mouse A9 cells and HFL-1 cells were trypsinized, they were mixed in a quarter to a half each, seeded in a 100 mm culture subculture with DMEM supplemented with 10% bovine fetal serum (FBS) and 15% bovine Fetal serum (FBS) was cultured in an equal volume of F12 for half a day to one day. Cell fusion follows the method described in (Shimizu et al., Handbook of Cellular Engineering, Yangtusha, pl27-, 1992). After washing the cell surface 2 times with DMEM, 2 milli-27. This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1223585 A7 B7 V. Description of the invention (25) Litre of PEG (1: 1.4 ) Solution treatment for 1 minute, and then replaced with 2 ml of PEG (1: 3) solution for 1 minute. The PEG solution was aspirated, washed three times with serum-free medium (DMEM), and then cultured in a normal medium (10% FBS, DMEM) for one day. Cells were trypsinized and dispersed, suspended in a dual selection medium (10% FBS, DMEM) containing ubaine (lxl (T5M, Xi Goma) and G4 18 (800 μg / ml) and seeded in 3 100 mm petri dish. After about 3 weeks of culture, the resulting colonies were dispersed with trypsin treatment and cultured in selective medium (10% FBS, DMEM) containing G41 8 (800 μg / ml). The cells were treated with trypsin Disperse, combine the 2 groups into one, and culture in 6 25 cm 2 centrifuge bottles (Kosta (Xiyi), 3025) until the cell density is 70 ~ 80% saturation, and replace with bile-containing Culture medium (20% FBS, DMEM) of rice (mouth small Qisanbu) (0.05 μg / ml, decarbonylated colchicine, Wako Pure Medicine) was cultured for 2 days to form microcells. After removing the culture solution, the Pre-incubated (37 ° C) solution of cytochalasin B (10 μg / ml, Xi Goma) filled the centrifuge bottle, insert the centrifuge bottle into a centrifugal container made of acrylic, performed at 34 ° C, 8000 rpm Centrifuge for 1 hour. The microcells were suspended in serum-free medium, and purified by filtration through a filter. Murine A9 cells were cultured in a 25-cm square bottle with 80% saturation, and the refined micronucleus was added and fused with PEG solution. Cultured in a selective medium containing G418 to isolate the community. Human chromosomes of each asexual line (2, 4th, 14th, 22nd) The following identification. All experimental operations and reagents except the above are in accordance with &lt; Shimizu et al., Handbook of Cell Engineering, Yangtusha, p 12 7-&gt;. (1) PCR Analysis and culture of isolated cells, using pure genetic DNA isolation kits (Puregene -28- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm)

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Line 1223585 A7 B7 V. Description of the invention (26) DNA Isolation kit (Gentra System) extracts genomic DNA, uses this genomic DNA as a template, uses human chromosome-specific primers, and selects to retain the 2, 4, 14, 22 clones of human chromosomes. PCR amplification was performed using about 0.1 micrograms of genomic DNA in accordance with (Innis et al., PCR Experimental Operations, HBJ Publishing Bureau, 1991, Thermal Cycler GeneAmp 9600, Perkin-Elmer Corporation). Din aq polymerase was made by Perkin-Elmer, and the reaction conditions were cycled once at 94 ° C for 5 minutes, then denatured at 94 ° C for 15 seconds, and cooled back to 54 ~ 57 ° C for 15 seconds. Perform 35 cycles of elongation at 72 ° C for 20 seconds. Primers use genes present on each chromosome (O 'Brien, Genetic Maps, 6th edition, Book 5, Cold Spring Harbor Laboratory Press, 1993) and polymorphic markers (Polymorphic STS Primer Pair, BIOS Corporation; Weissenbach et al., Nature 359 : 794, 1992; Walter et al., Nature Genetics, 7:22, 1994, etc.). Gene primers are made based on the base sequences of databases such as GenBank and EMBL. The names of the polymorphic primers and the sequences of the gene primers are shown in the following examples for the respective chromosomes (2nd, 1st, 2nd, 6th, 14th, 9th, and 22nd embodiments) : Example 2). The chromosome 2 was identified using the following genetic markers and polymorphic markers (Polymorphic STS Primer Pair, BIOS Corporation; D2S207, D2S177, D2S156, D2S159 BIOS Corporation). C / c (immunoglobulin / c constant): 5’-TGGAAGGTGGATAACGCCCT _ (sequence number 1), 5,-TCATTCTCCTCCAACATTAGCA (sequence number 2)

FABP1 (Monthly Fatty Acid, Binding Protein-1 Liver): 5'-GCAATCGGTCTGCC -29- This paper size applies to Chinese National Standard (CNS) A4 (210 X 297 mm) 1223585 A7 B7 V. Description of the invention (27 GGAAGA (Order 歹 [No. 3), S.-TTGGATCACTTTGGACCCAG (Serial No. 4)

Vk3-2 (immunoglobulin / C variable): 5, -CTCTCCTGCAGGGCCAG TCA (sequence number 5), 5'-TGCTGATGGTGAGAGTGAACTC (sequence number 6)

Vkl-2 (immunoglobulin / c variable): 5f-AGTCAGGGCATTAGCAG-TGC (sequence number 7), 5,-GCTGCTGATGGTGAGAGTGT (sequence number 8) (2) Fluorescent spot hybridization (FISH) FISH analysis according to (Matsuhara et al. , FISH experiment plan, Xiu Runsha, 1994) were performed using human chromosome-specific probes (CHROMOSOME PAINTING SYSTEM, Cambio). For example, 'the asexual reproduction of chromosome 2 is retained, and 10 out of 26 groups (745 asexual reproduction lines) have more than one asexual reproduction line. Among them, those who were all positive for the second chromosome-specific primers were 5 asexual breeding lines. These asexual lines were analyzed by FISH. The FISH analysis was performed in accordance with the method described in (Matsuhara et al., FISH experiment plan, Xiu Runsha, 1994), using a human second chromosome-specific probe (CHROMOSOME PAINTING SYSTEM, CANBIO). The human second chromosome in all the primers of the positive cells was observed in an approximately complete shape. In only one part of the asexual breeders with primer-positive clones, smaller independent chromosomes of human chromosome 2 were observed in several clones. Cells of chromosomes (Figure 1). As shown in Figure 1, horizontal row -30- This paper size is suitable for S ® family scale (CNS) A4 (21〇 X 297mm) · Binding

Line 1223585 A7 B7 V. Description of the invention (28) List of asexual breeding line names, primers used in vertical list PCR. • Form positive, Form X negative. In addition, the existence pattern of human chromosome 2 observed by FISH is at the bottom. Not implemented in the recorder. Similarly, human A9 cells on chromosomes 4, 14, and 22 must be retained. (Example 2) The human chromosome 22 was introduced into mouse ES cells as a chromosome donor cell by the microcell method, and the mouse A9 cell line (hereinafter referred to as "human chromosome 22") retained in (Example 1) was used A9 / # 22). As a chromosomal receptor cell, a mouse ES cell line E14 (purchased from Martin L. Hooper, Hooper et al., Nature, 326: 292, 1987) was used. The culture method of E14 was in accordance with the method described in <Sangshin Shinichi, Biological Manipulation Series 8, Gene Target, Yangtusha, 1995 &gt;, and methymycin (^ 彳 卜 Y 彳 シ &gt;) C as a vegetative cell ( Yugema) treated G.418-resistant STO cell line (obtained from Osaka University, Professor Kondo Shouren). First, microcells were prepared from about 108 A9 / # 22 cells according to the method reported by Shimizu et al. (Handbook of Cell Engineering, Yangtusha, 1992). The entire amount of the obtained microcells was suspended in 5 ml of DMEM. About 107 E14 were dispersed with trypsin, washed three times with DMEM, suspended in 5 ml of DMEM, combined with microcells, and centrifuged at 1250 rpm for 10 minutes to remove the supernatant. Dissolve the precipitate thoroughly by vortex, add 1 丄 4 PEG solution (5 g PEG 1000, &lt; Wako Pure Chemicals &gt;, 1 ml 01 ^ 30 &lt; Xigoma &gt; dissolved in 6 ml 01 ^ £ 1 ^) 0.5 10 ml of DMEM was allowed to stand for 1 minute and 30 seconds at room temperature. Immediately centrifuge at 1250 rpm for 10 minutes to remove the supernatant. The pellet was suspended in 30 ml of ES cell culture medium, and then sown on 3 interspersed vegetative cells with a diameter of 100 mm -31. Specifications (210X297 mm)

Line 1223585 A7 B7 V. Description of Invention (29) The plastic culture union (Kolin) for tissue culture. After 24 hours, the medium was exchanged with 300 micrograms / ml of G418 (GENETICIN, Xi Goma), followed by daily medium exchange. After 1 week to 10 days, drug resistance clusters appeared. Its frequency is 0 to 5 per 107 E14 cells. The colonies were picked up and proliferated, and suspended in 1 ml of a storage medium (ES cell culture medium + 10% DMSO &lt; Xigoma &gt;) every 5 × 10 6 cells, and stored frozen at -80 ° C. At the same time, for each drug-resistant strain, genomic DNA was prepared from 106 to 107 cells by a pure gene DNA isolation kit (Gentra System). Fragmentation of human chromosome 22 is performed by irradiating microcells with r-rays (Koi et al., Science, 260: 361, 1993). The microcells obtained from about 108 A9 / # 22 were suspended in 5 ml of DMEM, and made of 7 cells 40 (Canadian Atomic Energy Corporation), and irradiated with 60 Gy ray (1.2 Gy / min x 50 min) on ice. R-ray-irradiated microcells were fused with non-irradiated microcells. As a result of the selection of drug-resistant strains, the frequency of drug-resistant strains was 1 to 7 out of 107 E14 cells. For drug-resistant strains, the situation Similarly, DNA was obtained by cryopreservation. Reserving the introduced chromosomes in the non-irradiated microcell drug-resistant strains E14 / # 22-9, Ε14 / # 22-10, and τ-ray-irradiated microcell drug-resistant strains E14 / # 22-14, E14 / # 22-25, by Check (1) to (3) below. (1) PCR analysis (Figure 2) Using the genomic DNA of the drug-resistant strain as a template, the gene (Geneiic Maps, preface) and polymorphic STS Primer Pair (Polymorphic STS Primer Pair) on the human chromosome 22 were detected by PCR. BIOS company: D22S315, D22S275, D22S278, D22S272, D22S274; Nature 359: 794, -32- This paper size applies to Chinese National Standard (CNS) A4 specifications (210 x 297 mm) 1223585 A7 B7 V. Description of the invention (30 1992 ). Write down the sequence of the gene primer oligonucleotide based on the base sequence obtained from the database of GenBank, EMBL, etc. PVALB (parvalbumin: 5 * -TGGTGGCTGAAAG CTAAGAA (SEQ ID NO: 9) , 5 丨 -CCAGAAGAATGGTGTCATTA (sequence number 10) MB (myoglobin): 5, -TCCAGGTTCTGCAGAGCAAG ((sequence number 11), 5, -TGTAGTTGGAGGCCATGTCC ((sequence ij number 12) D1A1 (cytochrome b-5 reductase) : 5'-CCCCACCCATGATCCAG TAC (Sequence 歹 J No. 13), 5,-GCCCTCAGAAGACGAAGCAG ((Serial Number 14)

Ig λ (immunoglobulin λ): 5,-GAGAGTTGCAGAAGGGGTGACT ((sequence number 15), 5, -GGAGACCACCAAACCCTCCAAA ((sequence number 16)) ARSA (aryl stone charged acid g per A): 5, -GGCTATGGGGACCTGGGCTG ((sequence number 17), 5'-CAGAGACACAGGGGCGCGAGAAG ((SEQ ID NO: 18) Using about 0.1 micrograms of genomic DNA as a template, PCR amplification was performed on the 10 types of primers described above (Innis et al., Previous note). All of the primers were isolated from T-rays, and amplified products of the expected length were detected for some of the primers. The above results are shown in Figure 2. In Figure 2, the left side is based on the human chromosome 22 Schematic chromosome map of the G-band image and several markers for unknown locations showing where they are located (〇 'Brien, GENETIC MAPS, 6th edition, BOOK 5, etc.). Gene and polymorphisms are arranged redundantly Method 'is based on the information available so far (Science, -33- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm).

Line 1223585 A7 B7 V. Description of the invention (31 HUMAN GENETIC MAP, 1994, Nature Genetics, 7:22, 1994, Nature 3 59: 794, 1992, etc.) 'shows approximate positional relationship' so the order may not be correct. For the four types of G418-resistant E14 cell lines, the expected amplified product detected by PCR is indicated by _, the undetected indicated by □, and the observation results by FISH analysis are shown on the lower side. A9 / # 22 is the chromosome donor cell. (2) Southern aspiration analysis. Southern aspiration analysis analyzed the L1 sequence of human-specific repeated sequences (104 to 105 replicas per haploid genome, obtained from RIKEN DNA Bank, Nucleic acids research, 13; 7813, In 1985, a 1.4 kb EcoRI-BamHI fragment from pUK19A was used as a probe. For about 2 micrograms of genomic DNA treated with restriction enzymes (Bglll, manufactured by Takara Shuzo), according to Ausubel et al., Current Protocols in Molecular Biology, John Wiley & amp The method described in Sons, Inc., 1994 &gt; was performed. As a result, human L1 sequences and hybridized loops were detected in most of the drug-resistant strain DNA. For the two unirradiated strains, the pattern and the ratio of the amount of human chromosomal DNA to mouse genomic DNA that can be estimated from the concentration of each ring zone were the same as those of A9 / # 22. When the signal intensity of the entire 7-ray irradiated strain was compared with A9 / # 22, it was related to the degree of deletion shown by PCR analysis. (3) Fluorescent spot hybridization (FISH) FISH analysis is based on the method described in (Matsuhara et al., FISH experimental plan, Xiu Runsha, 1994), using the human chromosome 22 specific probe (CHROMOSOME PAINTING SYSTEM , Cambio). As a result, E14 / # 22-9 was transferred to the small part of most of the observed split images. -34- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm).

Line 1223585 A7 B7 V. Description of the invention (32) The form of mouse chromosome, the other 3 strains are used as independent chromosomes, and human chromosome 22 is detected. From the above experiments, the G418-resistant strains E14 / # 22-9 and E14 / # 22-10 were confirmed to retain all or most of the human chromosome 22, and E14 / # 22-14 and E14 / # 22-25 were confirmed to be retained A fragment of it. (Example 3) Techniques for obtaining, cultivating, injecting ES cell embryos into chimeric mice from ES cells retaining human chromosome 22, and transplanting them into temporary parental uterus, etc. Xiang Yan Shinichi, Biological Manipulation Series 8, Gene Target, Yangtusha, 1995 &gt; The G41 8-resistant ES cell line E14 / # 22-9 confirmed to be retained on the human chromosome 22 obtained in (Example 2) was obtained from frozen stock, and was used in C57BL / 6 X C3H F1 male mice (Japanese 10 to 15 embryos were injected into placental embryos obtained by mating with C3H male mice (Benkeria). About 2 to 5 days after the treatment of pseudopregnancy, about 10 embryos injected with ES cells were transplanted into the uterus of the parental ICR mice (Keliya Co., Ltd.) per unilateral uterus. The results are shown in Table 1. Table 1. Chimeric mouse ES cell line produced from E14 cell line that retains human chromosome 22 (fragment) / G418 Chimeric tolerant ES cell-infused mouse 'Contribution to hair color Human chromosome strain number Placental cell stage embryo number Number of mice ~ 10% 10-30% 30 ° / ^ Ε14 / # 22 9 166 29 16 7 3 6 As a result of transplanting 166 embryos, 29 child mice were born. Mosaic pieces -35- This paper size applies to China National Standard (CNS) Α4 size (210X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (33) The body is determined from the wild color (strong tea) from the host embryo, and whether the thin gray hair color from E14 cells is confirmed. Of the 29 births, the hair color was apparently a thin gray part, that is, there were 16 individuals whose contribution to E14 cells could be confirmed. The highest contribution rate is about 40% at K22-22. From this result, the mouse ES cell line E14 / # 22-9, which retains the human chromosome 22, confirmed the chimerism, that is, the ability to differentiate in the normal tissues of the individual mouse. (Example 4) Confirmation of the retention of human chromosomal DNA in various tissues of chimeric mice from ES cells retaining human chromosome 22. In addition to the determination of the coat color of Example 3, the retention of the introduced chromosomes was confirmed by PCR analysis using genomic DNA prepared from the tail as a template. Chimeric mice more than 3 weeks after birth, obtained tails according to the method described in "Katsuki Motoya, engineering experiment operation, Kodan Social Science, 1987", using pure gene DNA isolation kits, extracted Genomic DNA. Using the genomic DNA as a template, PVALB and D22S278 in the polymorphic primers used in Example 2 were used to confirm the amplification products. As a result of analyzing only 10 of the individuals whose hair color can be seen as a contribution, in all the individuals, at least a few amplification products by primers can be confirmed. The analysis of the southern sucking method was performed in the same manner as in (Example 2). The human L 1 sequence was used to probe 2 micrograms of tail genomic DNA from chimeric mice of 6 individuals and non-chimeric mice of 1 individual. As a result, in all chimeric individuals, the existence of most human L1 sequences can be confirmed, and its pattern is similar to E14 / # 22-9. The ratio of the mouse genome to the largest is about 10% (Figure 3). In Figure 3, Bglll digested 2 micrograms of genomic DNA was used in each lane. Marked with 32P -36- This paper size applies Chinese National Standard (CNS) A4 (210X297mm) binding

Line 1223585 A7 B7 V. Description of the invention (34) The L1 sequence of the number of people is a probe, and the signal is detected by the image analyzer-BAS2000 (Fuji Photograph (7 彳 A A)). From the right, the genomic DNA and control DNA from the tail of the chimeric mouse (feet 22-6, 7, 8, 9, 10, 11, 12: 9 are non-chimeric individuals) (C: C is the E14 / # 22-9 and E14 genomic DNA mixed with a weight ratio of 1: 9). The molecular weight of DNA is shown on the left, and the chimeric rate of each chimeric individual (-: 0%, +: ~ 10%, ++: 10 ~ 30%) is shown on the right. K22-7), The genomic DNA was obtained from the brain, liver, muscle, heart, spleen, thymus, ovary, and kidney by ISOGEN (Japanese company), and the genes used in (Example 2) were used for each tissue. MB and DIA1 in the library are analyzed by PCR. As a result, both primers were found in all tissues, and the expected expansion product was confirmed. The results with the DIA1 primer are shown in Figure 4. After electrophoresis on 2% agarose gel, PCR products were detected by ethyl bromide staining. Each path in Figure 4 is from the left. B: Brain, L: Liver, SM · Skeletal muscle, Η: Heart, Sp: Spleen, Th: Thymus, Ον: Nest, K: Kidney, nc: Non-chimeric Mouse tail DNA (negative control group), pc: human fibroblast (HFL-1) DNA (positive control group). From these results, it was confirmed that E14 / # 22-9 contributes to various normal tissues in mouse individuals and retains human chromosome 22. (Example 5) The expression of human genes in chimeric mice from ES cells retaining human chromosome 22 was used as a sample for confirming the expression of human genes. Individuals whose contribution to hair color was 5% (K22-7 ) Tail, crushed after freezing in liquid nitrogen. This is a mixture of skin, bone, muscle, blood and other tissues. Since then -37- This paper size applies to China National Standard (CNS) A4 (210X 297mm) binding

Line 1223585 A7 B7 V. Description of the invention (35 Total RNA was extracted by ISOGEN (Japanese company), and mRNA of human myoglobin (MB) and human cytochrome b5 reductase (DIA1) was detected by RT-PCR method. RT-PCR was performed according to the method described in &lt; Innis et al., PCR experimental operation, HBJ Publishing Bureau, 1991 &gt;. Reverse transcription application primers used random hexamer oligonucleotide (final concentration 100 picomolar, (Made by Takara Shuzo), and the reverse transcriptase was made by BRL (superscript). Note the primers using cDNA as a template for amplification. MB: 5, -TTAAGGGTCACCCAGAGACT ((Sequence 歹 | J No. 19), 5, -TGTAGTTGGAGGCCATGTCC ( (SEQ ID NO: 20) DIA1: 5, -CAAAAAGTCCAACCCTATCA ((SEQ ID NO: 21), 5, -GCCCTCAGAAGACGAAGCAG (SEQ ID NO: 22) As a result, mRNA-specific amplification products of the two genes were detected (Figure 5) RT-PCR products were electrophoresed on 2% agarose, and detected by ethidium bromide staining. In Figure 5, the M table is labeled (Hindlll digested into DNA + HaeIII digested p X174DNA, Takara Shuzo), MB represents human myoglobin, DIA1 represents human cytochrome b5 reductase, WT represents wild-type C3H small In addition, for the same individual (K22-7), the total RNA was extracted from the brain, heart, thymus, liver, spleen, kidney, ovary, and skeletal muscle using ISOGEN, and RT-PCR of each organ was performed by using the two primers described above. As a result, DIA1 is found in all organs, and MB only confirms the expected expansion products in the heart and skeletal muscle (Figure 6). It is known that myoglobin expresses specifically in muscle cells (Bassel-Duby et al., MCB, 12: 5024, 1992). This result indicates that genes on the human chromosome can be controlled by normal tissue-specific expression in mouse individuals. PCR products were detected by 2% agarose electrophoresis and stained with ethidium bromide. Figure 6, each -38- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (36) Road from left Table B: Brain, Η: Heart, Th: Thymus, L: Liver, Sp: Spleen, K: Kidney, Ov: SM, Skeletal Muscle, M: Mark (above). The lower band observed in the MB results is considered to be a non-specific product. That is, it was confirmed that the introduced human chromosome 22 functions in the normal tissues of the chimeric mouse. (Example 6) A human chromosome 4 or a fragment thereof is introduced into ES cells as a chromosome donor cell, and a mouse A9 cell strain (hereinafter referred to as A9 / #) retained on the human chromosome 4 obtained in (Example 1) was used. 4). As a chromosome receptor cell, a mouse ES cell line E14 was used (the same as in Example 2). Microcell fusion experiments and selection of G4 18 resistant strains were performed in the same manner as in (Example 2). The frequency of drug-resistant strains was 1 to 2 per 107 E14 cells. The genomic DNA was freeze-preserved for the drug-resistant strain in the same manner as in (Example 2). The preservation of human fourth chromosomes or fragments thereof in the drug-resistant strains E14 / # 4-4, E14 / # 4-7, and E14 # 4-ll was confirmed by (1) to (3) below. (1) PCR analysis (Figure 7) Using the genomic DNA of a drug-resistant strain as a template, the gene existing on human chromosome 4 (0 · Brien, Genetic Maps, 6th edition, Book 5, Cold Spring Harbor) was detected by PCR. Laboratory Press, 1993) and polymorphic markers (Polymorphic STS Primer Pair BIOS Company: D4S395, D4S412, D4S422, D4S413, D4S418, D4S426, Fll; Nature 359: 794, 1992). Make a note of the sequence of the gene primer oligonucleotide fenamic acid made based on the base sequence obtained from the databases of GenBank, EMBL, etc. -39-This paper size applies to China National Standard (CNS) A4 (210 X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (37 HD (Huntington disease): 5'-TCGTTCCTGTCGAGGATG AA ((sequence ij number 23), 5f-TCACTCCGAAGCTGCCTTTC ((sequence number 24) IL-2 (Interleukin) -2) 5'-ATGTACAGGATGCAACTCCTG ((serial number 25), 5f-TCATCTGTAAATCCAGCAGT ((serial number 26) KIT (c-set)) 5f-GATCCCATCGCAGCTCTACCGC ((serial number 27), 5'-TTCGCCGAGTAGTCGCACGG (( SEQ ID NO: 28) FABP2 (fatty acid binding protein 2, intestinal): 5f-GATGAACTAGTCCA GGTGAGTT ((SEQ ID NO: 29), 5'- CCTTTTGGCTTCTACTCCTT CA ((SEQ ID NO: 30)) PCR amplification results of the 11 types of primers described above For all or a part of the primers of 3 strains, confirm the expected amplification product. For E14 / # '4, E14 / # 4-7 strains, there are some missing areas. The above results are shown in Figure 7 On the left side of FIG. 7 is a schematic chromosome map based on the g-band image of the human chromosome 4, and where the markers are located, where are they located (see Example 2). Gene and polymorphism markers The arrangement is based on The information available today (refer to Example 2) indicates the approximate positional relationship, and the order may not be correct. For the three types of G418-resistant E14 cell lines, the expected amplified product label was detected by PCR, and there was no detection. The target 1 has been indicated by □. The observation results obtained by FISH analysis are shown below. 8 9 / # 4 Chromosome donor cells. (2) Analysis by Southern absorption method (Figure 8)-Analysis by Southern absorption method Similar to (Example 2), the genomic DNA obtained from Ei4 / # 4-4 and E14 / # 4-7 was performed using the human L1 sequence as a probe. -40- This paper standard applies Chinese National Standard (CNS) A4 size (210X 297mm) binding

Line 1223585 A7 B7 V. Description of the invention (38) As a result, the majority of the two strains detected a loop hybridized to the human L1 sequence. Compared with A9 / # 4, the overall signal strength is related to the degree of loss as shown by PCR analysis. In Figure 8, 2 micrograms of genomic DNA disappeared from Bglll for each lane. A 32P-labeled human L 1 sequence was used as a probe, and the signal was detected by the image analyzer BAS2000 (Fuji Photo-Wim Company). In Figure 8, each channel is from the left table 1: Α9 / # 4 (chromosome donor cells), 2: A9 / # 4 + A9 (1: 2), 3: A9 / # 4 + A9 (1: 9) , 4: A9, 5: E14 / # 4-7, 6: E14 / # 4-4. 2, 3 are two kinds of DNA mixed in the ratio shown in parentheses. DNA molecules on the left table 〇 (3) Fluorescent spot hybridization (FISH) FISH analysis was performed in the same manner as in Example 2 using a human chromosome 4 specific probe (CHROMOSOME PAINTING SYSTEM, Cambio). As a result, the human fourth chromosome or a partial fragment was detected in most of the three split images. E14 / # 4-4 was transferred to mouse chromosomes, and the other two strains existed on separate chromosomes. The relative size of the observed human chromosomes was consistent with what was speculated from the results of the PCR analysis. From the above experiments, it became clear that the obtained G4 18 resistant strain retains all or part of the human chromosome 4. (Example 7) Production of chimeric mice from ES cells retaining partial fragments of human chromosome 4 G418-resistant ES cell lines E14 / # 4-4, El4 / # 4-7, 10 to 15 embryos were injected into placental embryos obtained in the same manner as in Example 3. Pseudopregnancy treatment Child daughters of the parental ICR mice (Japan Crelia) on 2.5 days, each -41-This paper size applies to China National Standard (CNS) A4 (210X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (39) About 10 ES cells were transplanted into the embryo from a unilateral uterus. The results are shown in (Table 2). Table 2. Chimeric mouse ES cell line made from human E4 cell line that retains human chromosome 4 (fragment) Number of rats ~ 10% 10-30% 30% ~ Ε14 / # 4 4 160 8 5 5--7 80 5 2 1 1-As a result of 240 implanted embryos, 13 child mice were born. The chimeric individuals were judged by whether there was a light gray hair color from E14 cells in the wild color (strong tea) from the host embryo. Of the 13 births, the hair color was obviously light gray, and there were 7 individuals who could confirm the contribution of E14 cells. In addition, the highest contribution rate among individuals from E14 / # 4-7 is about 15%. From this result, it was confirmed that the mouse ES cell lines E14 / # 4-4 and E14 / # 4-7 retaining the human chromosome 4 fragment retain the chimeric forming ability, that is, the ability to differentiate into normal tissues of the mouse individual . (Example 8) Confirmation of the retained chromosomal DNA and expression of G4 18 tolerance factor in chimeric mice from which the human fourth chromosome fragment was retained (1) PCR analysis was performed on the chimeric-mouse obtained from (Example 7) One of E14 / # 4-7 (K # 4-7-l: Chimeric rate of about 5%), one of E14 / # 4-4 (K # 4-4-41: Embedded (Combination rate is about 5%), as in (Example 4), the base is prepared from the tail-42- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) 1223585

Due to group DNA. Using this as a template, the polymorphic marker F 11 detected by E14 / # 4-7 and E14 / # 4_4 in the fourth chromosome analysis primer shown in (Example 6) was analyzed by PCR. As a result, expected amplification products were detected in both individuals. (2) Analysis of the Southern Suction Method (Figure 9) The analysis of the Southern Suction Method is the same as in (Example 2), for individuals from E14 / # '(K # 4-7-1: Chimerism rate is about 5%) The human L1 sequence was used as a probe to perform 2 micrograms of genomic DNA from the tail. As a result, most of them confirmed the existence of the human L1 sequence, and its pattern was similar to ει4 / # 4 · 7. The ratio of mouse to mouse genome is about 10% of E14 / # 4-7. In Figure 9, each lane uses a genome of 2 micrograms of tail from Bglll digestion, ONA. The 32P-labeled human L1 sequence was used as a probe, and the signal was detected by the image analyzer BAS2000 (Fuji PhotoHume Corporation). The molecular weight of DNA is shown on the left. Each lane is from the left. Table 1: 7-1, 2: Blank, 3: Ε14 / # 4-7. (3) In the G418 tolerance test chimeric mouse derived from fibroblasts from the tail, for one individual from E14 / # 4-7 (κ # 4-7-1: interference rate about 5%), from E14 One individual of ## 4-4 (κ # 4-4-41: Chimerism rate of about 5 ° / °) 'Fibroblasts were prepared from the tail as follows. 〇να Preparation was the same as in (Example 4). The tail of the chimeric mouse was cut to 5 mm to 10 mm, washed several times with PBS / lmM EDTA, and an incision was made with a surgical knife. Cut the internal tissue cells. The tissue pieces were transferred to a tube containing 5 ml of PBS / lmM DTA, and left at room temperature for 30 minutes to 1 hour. After that, leave 1 liter of PBS / EDTA, remove the supernatant, add 1 ml of 0.25% trypsin 'and vortex at room temperature for 5 ~ 10 minutes or suck and release the tissue at the same time with a pipette -43- Zhang scale is applicable to China National Standard (CNS) A4 (210X297 mm)

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Line 1223585 A7 _ ___B7 V. Description of the invention (41) Unlock. Centrifuge at 1000 rprn for 10 minutes. Shen Dian was suspended in 2 ml of DMEM (10% FCS) and seeded in a 35 mm petri dish. After 7 to 10 days of culture, the cells were stripped from the Petri dish by membrane protease treatment, and about 104 cells per culture were seeded in two 35 mm Petri dishes, one of which was added with a final concentration of 400 彳Masako / ¾ liter G418 'culture for 5-7 days, observe individual cultured jni cells. Under these conditions, about 100% of fibroblasts from wild-type ICR mice died in the presence of G418. As a result, it was confirmed that both individuals had G418-resistant fibroblasts. From these results, 'E14 / # 4-7 and E14 / # 4-4 were confirmed in individual mice, contributing to the normal tissues of various species, and retaining the human chromosome 4 fragment. (Example 9) Human ES chromosome 14 or a fragment thereof was introduced into mouse ES cells as chromosome donor cells. The mouse A9 cell line (hereinafter referred to as A9 / #) of human chromosome 14 obtained by retaining (example 丨) 14). As a chromosome receptor cell, a mouse ES cell line TT2 (purchased from (Biotech Orient (currently 47, 7 and 7), Yagi et al., Analystiicai Biochem., 214: 70, 1993) was used. The culture method of TT2 was according to the method described in <Shan Han Shinichi, Biological Manipulation Series 8, Gene Target, Yangtusha, 1995>, and the vegetative cells were treated with maltomycin c (Xigoma). =: Subculture cells (purchased from Life Technology Oriental Co., Ltd.) 'Microcell fusion experiments and selection of G41 8-resistant strains were performed in the same manner as in (Example 2). 3 to 6. Frozen preservation and genomic DNA acquisition were performed in the same manner as in (Example 2). Fragmentation of the flying human chromosome 4 was performed by irradiating 7 cells with 7 rays (Koi et al., Science, 260: 361, 1993). Approx. 108 &gt; Shanding 1U <obtained from A9 / # l4 -44-

1223585 A7 B7 V. Description of the invention (42) The microcells are suspended in 5 liters of DMEM, and r cells 40 (previous note) are irradiated with 30Gyi 7 shots on ice (1.2Gy / min X 25min). Microwave cells irradiated with τ rays were fused with non-irradiated microcells. As a result of selection of the drug-resistant strains, the frequency of the drug-resistant strains was 3 per 107 TT2 cells. The drug-resistant strain was frozen and stored in the same manner as in (Example 2), and DNA was obtained. 4 humans including G41 8-resistant strains 1-4, 1-5 that have been transplanted with micro-cells that have not been irradiated with 7 lines, 4-1, 3-2 that have been transplanted with micro-cells that are irradiated with r-rays (30 Gy) The reservation of the chromosome 14 or part of the fragment is confirmed by (1) and (2) below. (1) PCR analysis (Fig. 10) Using the genomic DNA of a drug-resistant strain as a template, the gene present on human chromosome 14 (〇, Brien, Genetic Maps, 6th edition, Book 5, Cold Spring) was detected by PCR. Harbor Laboratory Press, 1993) and Polymorphic STS Primer Pair BIOS Company: D14S43 'D14S51, D14S62, D14S65, D14S67, D14S72, D14S75' D14S78, D14S81, PCI; Nature 359: 794, 1992; Nature Genetics, 7 : 22, 1994). Write down the sequence of the gene primer oligonucleotide based on the base sequence obtained from the database of GenBank, EMBL, etc. NP (nucleus: y: phosphate 4 g g): 5, -ATAGAGGGTACCCACTCTGG ((sequence number 31), 5, -aaccaggtAggttgatatgg ((sequence number 32)

TCRA (T-cell receptor a): 5, -AAGTTCCTGTGATGTCAAGC -45- This paper size applies to China National Standard (CNS) A4 (210X297 mm)

Line 1223585 A7 B7 V. Description of the Invention (43) ((Serial Number 33), 5, -TCATGAGCAGATTAAACCCG ((Sequence 歹 J # 34))

MYH6 (Heart Myosin Heavy Chain): 5 * -TGTGAAGGAGGACCA

GGTGT ((sequence number 35), 5, -TGTAGGGGTTGACAGTGACA ((sequence number 36) IGA2 (immunoglobulin alpha-2 constant) · 5, -CTGAGAGATGCCTCTG GTGC ((sequence number U) 37), 5, -GGCGGTTAGTGGGGTCTTCA ( (SEQ ID NO: 38) IGG1 (immunoglobulin &quot; I constant): 5'-GGTGTCGTGGAACTCA GGCG ((SEQ ID NO: 39), 5, -CTGGTGCAGGACGGTGAGGA ((SEQ ID NO: 40) IGM (immunoglobulin # constant): 5, -GCATCCTGACCGTGTCC GAA ((sequence number 41), 5, -GGGTCAGTAGCAGGTGCCAG ((sequence number 42) IGVH3 (immunoglobulin repeat variable -3): 5, -AGTGAGATAAGCAGT GGATG ((sequence number, j number 43)), 5, -GTTGTGCTACTCCCATCACT ((SEQ ID NO: 44) Using the genomic DNA of 4 drug-resistant strains as a template, the results of PCR amplification of the 18 types of primers described above were performed in the same manner as in (Example 2), and all or a part of the primers were confirmed as the expected amplification products. The micro-cells irradiated with T-rays confirmed that the obtained drug-resistant strains, 3-2, had a tendency to delete a part of chromosome 14. Moreover, in the case of using non-irradiated micro-cells, the deletion was also seen as in 1-4 strains. The above The results are shown in Section 10 Figure. In Figure 10, the left side shows a map of a modular chromosome based on the G-band image of human chromosome 14, and it is -46-this paper is suitable for financial countries @ 家 榡 准 (CNS) A4 size_x297 公 爱)

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Line 1223585 A7 B7 V. Description of the invention (44) Where are the several markers that are clearly understood on the location of the endless belt (see Example 2). The arrangement method of the gene and polymorphism markers is based on the information available so far (refer to Example 2) and shows approximate positional relationships, so the order may not be correct. For the four types of G418-resistant TT2 cell lines, the markers of the expected amplified products were detected by PCR, and the markers not detected were indicated by □. A9 / # 14 is the chromosome donor cell. The right end shows the results of Example 11 (1). (2) Fluorescence on-site hybridization (FISH) FISH analysis according to (Matsuhara et al., FISH experiment plan, Xiu Runsha, 1994) The method described in the following uses the human chromosome 14 specific probe (CHROMOSOME PAINTING SYSTEM, Cambio) )get on. As a result, in most of the four isolates, the human chromosome 14 or a partial fragment was observed as an independent chromosome. The relative size of the observed human chromosomes is consistent with that predicted from the results of PCR analysis. From the above examples, it can be confirmed that the obtained G418-resistant strains 1-4, 1-5, 3-1, and 3-2 retain all or part of the human chromosome 14. (Example 10) Four chimera-resistant ES cell lines retaining human chromosome 14 (1- (4, 3-1, 3-2, 1-5). Into eight cell-stage embryos obtained by mating male and female mice by ICR or MCH (ICR) (Kelia, Japan), each embryo was injected with 8 ~10. The cells were cultured overnight in ES medium (Example 9), after they occurred in placental cells, and were lent to the uterus of parental ICR mice (Japan_Kelia) 2.5 days after pseudopregnancy treatment. About 10 injected embryos were transplanted. The results are shown in (Table 3). Table 3. Production of TT2 cell line from human chromosome 14 (fragment) reserved -47- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1223585 A7 B7 V. Description of invention (45) Murine ES cell line / G418-resistant ES cell-injected children Chimeric mice Contributing rate to coat color Human chromosome strain No. 8 Cell stage embryo number Number of rats ~ 20% 20-50% 50-80% TT2 / # 14 1-4 98 20 1--1 1-5 110 14 2 1-1 3-1 103 11 2 1 1-3-2 183 19 3 to 2 1 Mice. Chimeric individuals are judged by whether the white color from the host embryo (ICR) can be identified as the hair color of wild color (strong tea) from TT2 cells. Of the 64 animals that were born, there was obviously a wild-colored part, and it was confirmed that there were 8 individuals whose ES cells contributed. The highest contribution rate is about 80% of one individual from 1-4. From this result, the G4 18-resistant ES cell line (1-4, 1-5, 3-1, 3-2) that retains the human chromosome 14 or a fragment thereof was confirmed to retain the chimera-like energy reduction, that is, to differentiate into Ability of normal tissues in individual mice. (Example 11) The retention of human 14th chromosome fragments in chimeric mice from ES cells retaining human 14th chromosome fragments was confirmed in the human 14th partial chromosome fragments of chimeric mice obtained in (Example 10) Reservation, confirm by (1) ~ (3) below. (1) PCR analysis using DNA from various tissues The same as in (Example 4) for one individual from 3-1 (K3-1-1; chimerism rate of about 25%) in the chimera , Genomic DNA was prepared from the tail. With -48- This paper size applies Chinese National Standard (CNS) A4 (210X 297mm) binding

Line 1223585 A7 B7 V. Description of the invention (46) This is a template, and PCR analysis is performed on all 14 kinds detected by the 14th chromosome analysis primer 3-1 shown in (Example 9). As a result, the expected amplification products were detected for all 14 types (Figure 10). In addition, for the same individual (K3-1-1), genomic DNA was obtained from the brain, kidney, spleen, heart, liver, and thymus by using pure genetic DNA isolation kits, and IGM primers were used for each tissue ( Example 9) PCR analysis. As a result, the expected amplification product was confirmed in all the tissues (Fig. 11). The PCR products were detected by 2% agarose gel electrophoresis and stained with ethidium bromide. In Figure 11, each channel is from the left. Table B: Brain, K: Kidney, Sp: Spleen, Η · · Heart, L: Liver, Th · Thymus, pc · · Human Fibroblast (HFL-1) DNA ( Positive control group), nc: non-chimeric mouse tail DNA (negative control group), M: labeling (Hindlll digested into DNA + HaeIII digested pX174DNA, Takara Shuzo). (2) Among the G4 18 resistance test chimeric mice derived from tail fibroblasts, for 2 individuals from 3-2 (K3-2-1; chimerism rate of about 25%, K3-2-3; The chimerism rate was about 50%), from one of 1-4 individuals (K1-4-1; the chimerism rate was about 80%), and fibroblasts were prepared from the tail as follows. DNA preparation was the same as (Example 4). The tails of chimeric mice of about 3 to 6 weeks of age were cut into 5 mm to 10 mm. After washing several times with PBS / lmM EDTA, all the cuts were cut with a surgical knife to remove the cuticle The internal tissue is cut with a scalpel. The tissue pieces were transferred to a tube containing 5 ml of PBS / lmM EDTA, and allowed to stand at room temperature for 30 minutes to 1 hour. After that, 1 ml of PBS7EDTA was left, the supernatant was removed, 1 ml of 0.25% trypsin / PBS was added, and the tissue was completely unraveled while vortexing or sucking with a pipette at room temperature for 5 to 10 minutes. Centrifuge at 1000 rpm for 10 minutes, -49- This paper size applies to China National Standard (CNS) A4 (210X297 mm) binding

Line 1223585

The pellet was suspended in 2 ml of DMEM (10% FCS) and seeded in a 35 亳 mila dish. After 7 to 10 days of culture, the cells were sub-exfoliated from the culture by trypsin treatment. Each dish was about 1 〇4 cells were seeded in 4 35 mm cells and 400 micrograms / ml of G418 was added to 2 of them, and cultured for 5 to 7 days. Number: the number of cells in other culture dishes. Under this condition, about 100% of the fibroblasts from wild-type ICR mice died in the presence of G418 relative to the number of non-selective culture cells and the number of viable cells in the selection medium. The cell proliferation rate is the same under the two conditions, which is reflected by the contribution rate of the fibroblast group from fibroblasts derived from the G41 8-resistant ES cell line. As a result, as shown in (Fig. 12), all three individuals could confirm the existence of G41 8-resistant fibroblasts. In Figure 12, the tolerance rates for the individual individuals are averaged from the values of the selected / non-selected 35 mm Petri dishes of the two groups. ICR means wild-type ICR mice. (3) FISH analysis of G418-resistant fibroblasts derived from the tail The same method as in (Example 2). The above-mentioned ^ resistant fibroblasts (from K3-2-3, K1-4-1) FISH analysis. The probes used were all humans extracted from HFL-1 cells (Example 丨) were labeled with FITC (Matsuhara et al.'S FISH experiment plan, Xiu Runsha, 1994). As a result, both individuals were in most of the division images Separate fragments of human staining were observed. From these results, the TT2 cell line retaining the human chromosome 14 partial fragment was isolated from the mouse, and it was confirmed that the normal tissues of various species were contributed, and the human chromosome 14 partial fragment was retained. -(Example 12) The human chromosome 2 fragment was introduced into ES cells as a chromosome donor cell, and the human (-50) obtained from the retention (Example 1) was used. This paper size applies the Chinese National Standard (CNS) A4 specification ( 210X297 mm) -----

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Yuan 1223585 A7 B7 V. Description of the invention (48) 2 mouse chromosome A9 cell W23 (hereinafter referred to as A9 / # 2 W23). As a chromosome receptor cell, a mouse ES cell line TT2 was used (Example 9). Microcell fusion experiments and selection of G4 18 resistant strains were performed in the same manner as in (Example 2). The frequency of drug-resistant strains is 1-3 per 107 TT2 cells. Cryopreservation of the drug-resistant strain, and genomic DNA acquisition were performed in the same manner as in (Example 2). The retention of the human chromosome 2 fragment in the drug-resistant strains 5-1, 5-2, and 5-3 is confirmed by (1) and (2) below. (1) PCR analysis Using genomic DNA of a drug-resistant strain as a template, the gene (Genetic Maps, preface) existing on human chromosome 2 was detected by PCR method to detect the chromosome donor cell A9 / # 2 W23 The existence of C / c, FABP1. As a result of PCR amplification of each primer, for both primers, 3 strains confirmed the expected amplification products. (2) Fluorescence field hybridization (FISH) FISH analysis was performed in the same manner as in Example 2 using a human second chromosome-specific probe (CHROMOSOME PAINTING SYSTEM, Cambio). As a result, in most of the division images of all three strains, the human chromosome 2 fragment was detected as an independent chromosome. Its size is the same as that observed by A9 / # 2 W23. From the above experiments, it was confirmed that the obtained G41 8-resistant strain retained the human second chromosome fragment. (Example 13) Chimeric mice made from ES cells that retain human second chromosome-fragment fragments were obtained from (Example 12) from frozen stocks and confirmed to retain human numbers 2 -51-This paper applies to China National Standard (CNS) A4 specification (210X 297 mm)

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Line 1223585 A7 B7 V. Description of the invention (49) Chromosome partial fragments of G418-resistant ES cell line 5-1, 8-cell stage embryos obtained by mating male and female mice using ICR or MCH (ICR) (Kelia, Japan) In each embryo, 10-12 were injected. Cultured in ES medium (Example 9) overnight. After placental blasts occurred, the uterus of parental ICR mice (Japan Crelia) was temporarily borrowed 2.5 days after the treatment of pseudopregnancy. About 10 unilateral uterine transplants were performed. Injection embryos. Results of mosaic production (Table 4). Table 4. Chimeric mouse ES cell line made from human TT2 cell line that retains human chromosome 2 (fragment) / G418 chimeric mice that are resistant to ES cell injection Contributing rate to coat color Human chromosome strain number 8 cell stage embryo number Number of rats ~ 20% 20-50% 50-80% TT2 / # 2 5A 264 51 18 7 5 6 (W23) As a result of 264 implanted embryos, 51 children were born. The chimeric individuals were judged by whether a wild color (strong tea) from TT2 cells was confirmed in the white color from the host embryo (ICR) in the hair color. There were 51 wild-colored parts in the birth of 51, that is, 18 individuals whose ES cell contribution can be confirmed, and the highest contribution rate was about 80%. From this result, it was confirmed that G4 18 resistant ES cells (5-1) that retain a partial fragment of the human second chromosome retain chimerism, that is, the ability to differentiate into normal tissues of mouse individuals. (Example 14) -52- The chimeric mouse serum introduced into the human chromosome 14 fragment was applied to the paper size of China National Standard (CNS) A4 (210 X 297 mm).

Line 1223585 A7 B7 V. Explanation of the invention (50) Detection of human antibody concentration in human antibody heavy chain using enzyme-linked immunoabsorbent assay method K &gt; TV Seven Seven) (ELISA) measurement. ELISA was performed according to the methods described below. Toyama · Anton and Monoclonal Antibody Antibody Experiments, Kodansha, 1987; Anjan Chiba, Introduction to Monoclonal Antibody Laboratory Experiments, Kodansha, 1991; Ishikawa, Super High Sensitive Enzyme Immunity Assays, Society Publishing Center, 1993; Ed Harlow and David Lane, Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; A. Doyle and JB Griffiths, Cell &amp; Tissue Culture: Laboratory Procedures, John Wiley &amp; Sons Ltd. , 1996. With reference to the methods described in these documents, the reaction time was measured according to the measurement system, and the improvement was performed at 4 ° C for two nights. The antibody or antigen to human immunoglobulins for measurement was diluted from 0.5 to 10 μg / ml (from 100 to 500 times), and the ELISA plate was coated at 4 ° C for two nights. Serum samples were measured, blocked, and samples and labeled antibodies were diluted using 5% mouse serum (Xigoma, M5905) in PBS. Hybridoma culture supernatants were measured using PBS with 1% bovine fetal serum. When the chimeric mouse serum was diluted 20-fold, it was diluted with PBS. After the coated plate was washed, the plate was blocked and washed for 1 hour or more, and then the sample was cultured for 30 minutes or more. After the plate was washed, the enzyme-labeled anti-human immunoglobulin antibody diluted 100 to 50,000 times was added. After culturing for more than 1 hour, the plate was washed and the matrix solution was used to make hair color. In addition, according to the measurement system, the biotin-labeled antibody was washed in the plate in substantially the same manner, and then the antibiotic-enzyme complex was added thereto, and after washing, the base solution was added. The absorbance was measured with a microplate reader (Biotechnology, EL312e). -53- This paper size applies to China National Standard (CNS) A4 (210X297mm) binding

Line 1223585 A7 __ B7 V. Description of the invention (51) Taken from chimeric mice (Example 10, K3-1-2, K3-2-2, K3-2-3) from 29th to 35th day after birth, with ELISA analysis. 50 mM bicarbonate buffer pH9.6 diluted anti-human IgM mouse single-source antibody (Xigoma, 16385) was spread on a 96-well microtiter plate to add mouse serum (Xigoma, M5905) PBS diluted serum samples. Then, after peroxidase-labeled anti-human IgM goat antibody (Tago, 2392) was cultured, enzyme activity was evaluated by adding an ABTS matrix (Kirkegaard &amp; Perry Laboratories, 506200) at 405 nm. Refined human IgM antibody (Organon technology, 6001-1590) or human IgG antibody (Xigoma, 14506) is used as the standard. The standard is stepwise dilution with mouse serum in PBS. For human IgG measurement, an anti-human IgG goat antibody (Xigoma, 13382) was fixed to the plate, and a human IgG goat antibody (Xigoma, A0170) labeled with peroxidase was detected. The results are shown in (Table 5). Both human IgM and IgG were detected. After the third, 27th, 34th, and 41th day after birth, in a chimeric mouse (Example 10, K3-1-1, K3-2-1) retaining human chromosome 14 fragments, 0.2 ml of human serum albumin (HSA, Shigoma, A3782) in PBS was mixed with 0.2 ml of adjuvant (MPL + TDM emulsion, RIBI Immunochem Research) (0.25 mg / ml). Subsequent mouse sera were also analyzed by ELISA. The results are shown in (Figures 13 and 14). As a result, the concentration of human antibodies in the serum of chimeric mice immunized with HSA increased after immunization. In individual K3-1-1, human IgM18 μg / ml and IgG2.6 μg / were detected in the serum on day 17 after immunization. Ml. In mouse sera newly introduced into human chromosomes; the potency of human antibodies is unintentional. Table 5. Human antibody concentration (ELISA) in chimeric mouse serum -54- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1223585 A7 B7 V. Description of the invention (52) Post-binding mouse IgG (Mg / L) IgM (mg / L) K3-1-2 0.37 3.7 K3-2-2 0.33 5.9 K3-2-3 0.51 3.4 (Example 15) Chimeric mice introduced from human chromosome 14 Hybridomas producing human antibody heavy chains were obtained from (Example 14) chimeric mice immunized with human albumin (K3-1-1, Example 14). The spleen was removed on the 44th day after birth and mixed with myeloma tumor cells. Cell fusion to make a hybridoma. The method for producing hybridomas is based on &lt; Anton, Introduction to Single-source Antibody Experiment Operation, Talking about Social Sciences, 1991 &gt; The method described, non-myeloma cell, using P3X63-Ag · 8. 653 (purchased by Daiyoshimoto Pharmaceutical) . The cells were dispersed in 10 96-well plates, and after 1 week of culture, the culture supernatant was analyzed by ELISA. · An ELISA method was used to fix a single-source anti-human IgM mouse antibody (Xigoma, 16385) in a plate, and the same procedure as in Example 14 was performed to obtain 6 positive asexual breeding lines. With HSA as the antigen and 50 mM carbonate-bicarbonate buffer pH 9.6 as a 5 microgram / ml solution, 100 microliters were injected into all the milk holes in the ELISA plate. Detection using peroxidase-labeled anti-human IgA + IgG + IgM sheep antibody (Kirkegaard &amp; Perry Laboratories, 04-10-17). One positive clone was confirmed in 10 plates. This asexual breeding line is in one of six human IgM positive asexual breeding lines. This asexual breeding line (h4B7) was cultured. The culture supernatant was diluted and HSA was used as the antigen. When performing an ELISA using a peroxidase-labeled anti-human IgM sheep antibody (Tago, 2392) as described above, the culture supernatant was confirmed液 之 稀 -55- This paper size applies to China National Standard (CNS) A4 (210X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (53) The increase in the release rate decreases the absorbance. On the one hand, Yu dilutes human IgM (Organon technology, 6001-1590) to a sample of 2 μg / ml by using medium, without dependence on the dilution rate and low absorbance. This suggests that the antibody produced by hybridoma H4B7 is an antibody specific for HSA (Figure 15). In Figure 15, the horizontal axis indicates the dilution rate of the culture supernatant sample, and the vertical axis indicates the absorbance at 400 nm. (Example 16) Re-labeling of pramycin resistance of a human second chromosome fragment labeled with G41 8 resistance will retain A9 cells (W23) of a human second chromosome fragment labeled with G4 18 resistance (see Example 1. Figure 1) Culture in a 100 mm petri dish containing G418 (800 μg / ml) in selection medium (10% FBS, DMEM). The pPGKPuro (available from WHITEHEAD INSTITUTE, Dr. Peter W. Laird) containing the ploromycin resistance gene was linearized with the restriction enzyme Sail (Takara Shuzo) before transfection. Cells were trypsinized, suspended in Dalbeco's phosphate buffered saline (PBS) to 5xl06 cells / ml, and subjected to electroporation using a gene pulser (Biaure) in the presence of 10 μg of DNA (see Example 1). With a capacity of 25 // F, an electric hole method of small millimeters with a thickness of 4 mm (Example 1) was used to print a voltage of 1000 V at room temperature. The cells treated with the electroporation method were seeded in 3 to 6 100-mm culture dishes. One day later, the medium was exchanged with a dual selection medium containing 10 µg / ml of polomycin (Xigoma, P-7255) and G418 (800 µg / ml). After 2 to 3 weeks, the 200 communities that were born were organized into one group. The three groups of these cells were cultured in 2 to 3 rows of 25 cm 2 bottles to form microcells, and mouse A9 cells cultured in 25 cm 2 bottles were fused in the same manner as in Example 1. Move to two 100 mm petri dishes and cultivate with a dual selection medium containing G4 18 and puramycin, three groups -56- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) Bookbinding

Line 1223585 A7 _______B7 V. Description of the Invention (54) Two clonal breeding lines with dual chemical resistance can be obtained from one group. Here, the asexual breeding line is highly likely to introduce a pramycin resistance marker into a human chromosome 2 fragment. (Example 17) The introduction of human chromosomes into ES chromosome-introduced human cells was doubled. ES cell lines (E14 / # 14-3 6) that retain human 14 chromosome fragments labeled with the G418 tolerance gene were added at high concentrations of G4. Asexual propagation lines of ES cells that have doubled human chromosomes are grown in a medium of 18 (Biological Operation Series 8, Gene Target, Yangtusha, 1995). The primary cells of G4 18-resistant mice (purchased from Dongfang Life Technology) were not treated with methicillin, but were seeded in 100-mm culture dishes as vegetative cells. E14 / # 14-36 was sown in this 100 mm petri dish and exchanged with a medium having a G418 concentration of 16 mg / ml half a day later. Exchange medium every 1 ~ 2 days! After a week, the concentration of G418 was 10 mg / ¾ liter and the culture was continued. 15 cultures were taken out of the born colony, and the human 14th specific probe (refer to Example 9) was used for FISH analysis. As a result, in eight asexual lines, human chromosome 14 fragments have doubled. (Example 18) Obtaining a mouse ES cell line that retains both the human second chromosome partial fragment and the 14th chromosome partial fragment. Asexual breeding lines with dual drug resistance obtained in (Example 16), PG1 was used as a micro Cell donor cells, wild type A9 cells were used as recipient cells, and microcellular transfer experiments were performed to confirm that the human chromosome 2 fragment, which retains PG 丨, was additionally labeled with the ploromycin resistance gene. The acquisition of microcells and fusion with A9 cells were performed in the same manner as in Example 丨. As a result, a total of 59 G41 8 resistant communities appeared in the microcell fusion 10-day dance. For these communities, the paper size applies to the Chinese National Standard (CNS) A4 (210X297 mm) 1223585 A7 B7. 5. Description of the invention (55) After changing the medium containing 8 μg / ml of ploromycin, When cultured for another 3 days, 45 (76%) communities survived. In the micro-cell method, since only one or a small number of chromosomes are transferred in one recipient cell, the two tolerance genes are simultaneously transferred at a high rate, which indicates that the human chromosome 2 fragment of the G418 tolerance marker of PG1 is retained. Marked by a ploromycin resistance gene. In addition, since each marker gene was detected on a partial fragment of the human second chromosome, p9 is used for A9 / # 2 W23 (G418 resistance only: Example 16) pSTneoB (Example 1) and pPGKPuro (Example 16) for PG1. FISH analysis of probes (Matsuhara et al., FISH experiment plan, Xiu Runshe, 1994). As a result, one signal was observed on each of the two sister chromosome segments of the human second chromosome fragment identified at A9 / # 2 W23 (Example 12), and two signals were counted, which indicates that pSTneoB was inserted into the second human One of the chromosome segments. In addition, four signals were observed on PG1, chromosomal fragments of the same size. Since pSTneoB and pPGKPuro had the same sequence on a part of the vector, pSTneoB was also detected on the pPGKPuro probe. That is, from the four signals observed by PG1, two signals from pSTneoB and the other two signals from pPGKPuro can be considered. From this result, a part of the human chromosome 2 fragment of PG1 was retained, and it was confirmed that there were markers derived from both G4 18 resistance and pramycin resistance. This PG1 cell line was used to obtain mouse ES cells that retained both human chromosome 2 and 14 chromosome fragments as chromosome donor cells. As a chromosome receptor cell, a G4 18-resistant TT2 cell line 1-4 in which a human 14th chromosome partial fragment has been retained (Example 9). Micro-cell fusion experiment and selection of pulomycin-resistant strains, 0.75 μg / ml -58- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (56 "Pramycin concentration, the other is the same as the case of the selection of G418-resistant strains of (Example 9). This result, the frequency of occurrence of pulomycin-resistant strains is 1 -4 cells have 3 to 7 cells per 107. These prosomycin-resistant strains also proliferated in the presence of 300 μg / ml of G41 8, so it was confirmed that G41 8 tolerance was also retained. Freezing of dual-agent-resistant strains Preservation and acquisition of the genome 0να were performed in the same manner as in Example 2. The human chromosome partial fragment and the human chromosome 14 partial fragment were retained. For the dual drug-resistant strains PG5, PG15, and PG16, the following (1 ), And confirm with 再 for PG15. (1) PCR analysis uses genomic DNA of a dual-drug-resistant strain as a template, among genes (Genetic Maps, preamble) existing on human chromosomes 2 and 14; The chromosome was confirmed to exist in (Example 12, A9 / # 2 W23), and the primers confirmed to exist in (Example 9; TT2 / # 14 1-4) on chromosome 14 were the results of PCR amplification. All primers, 3 strains confirmed Expected amplification product. (2) Fluorescent spot hybridization (FISH) FISH analysis was performed in the same way as in Example 11 using FITC to label human full-length DNA as a probe. As a result, most of the split images Confirm the human chromosome fragment of two sizes. The larger one is related to (Example 9; TT2 / # 14 1 -4) the partial fragment confirmed by the human 14th chromosome-specific probe, and the small one is related to (implementation Example 12; TT2 / # 2_ 5-1) Some fragments confirmed by human chromosome 2 specific probes are the same size. (Figure 16) The results are shown. The chromosomes with low luminosity in the figure are from mice. FITC's Fluorescence Luminosity -59- This paper size applies to China National Standard (CNS) Α4 specification (210X297 mm)

Line 1223585 A7 B7 V. Description of the invention (57) Two chromosome fragments of higher size (indicated by arrows) are from humans, and are considered to be the human chromosome 14 and 2 partial fragments. From the above experiments, it was confirmed that the obtained double-vesicular ES cell line retained the human chromosome partial fragment 2 and the 14 chromosome partial fragment. (Example 19) A chimeric mouse will be produced from a mouse ES cell line that retains both the human second chromosome partial fragment and the 14th chromosome partial fragment (obtained in Example 18, and it has been confirmed that the human second chromosome partial fragment and G41 of partial fragment of chromosome 14 8. Puramycin dual-resistant TT2 cell lines PG5, PG15, and PG16 are taken from frozen stocks and obtained by mating male and female mice of ICR or MCH (ICR) (Kelia, Japan) Eight-cell stage embryos were injected with 10-12 embryos each. The ES cells were cultured overnight in the medium (Example 9) to allow placental cells to develop, and the parental ICR mice were temporarily borrowed on the 25th day after pseudopregnancy treatment ( In the uterus of Japan, about 10 implanted embryos are transplanted from each unilateral uterus. The results of the chimera are shown in (Table 6). Table 6. Since the second human chromosome segment and the 14th chromosome are retained at the same time Fragmentation of a mouse ES cell line

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1223585 A7 B7 V. Description of the invention (58) ES cell line / double-resistant ES cell-inducing mouse chimera contributes hair color to human chromosome strain number 8 cell stage embryo number ~ 10% 10- 50% 50%, TT2 / PG5 160 26 8 7 1-# 14 + # 2 PG15 168 15 3 1 2-PG16 223 32 12 3 6 3 As a result of implanting 55 embryos, 73 child mice were born. The chimeric individuals were identified by the wild color (strong tea) from TT2 cells in the white color from the host embryo (ICR) due to whether the hair color was confirmed. Obviously, the 73 middle-breast color was born with a wild-colored part, which means that there were 23 individuals with ES cell contribution. From this result, it was confirmed that the ES cell lines (PG5, PG15, PG16) which retain the partial fragments of human chromosome 2 and partial fragments of chromosome 14 retain the chimeric forming ability, that is, the ability to differentiate into normal tissues of mouse individuals . (Example 20) Human antibodies were detected in chimeric mouse sera from E S cells that simultaneously retained human second chromosome partial fragments and fourteenth chromosome partial fragments. Among the chimeric mice produced in (Example 19), KPG-15 (9 weeks of age; from PG5, chimerism rate of 10%) and KPG-18 (5 weeks of age; from PG5, chimerism rate of 10%) 2), mix human serum albumin (HSA, Igoma, A3782) dissolved in PBS with an adjuvant (MPL + TDM emulsion, RIBI Immunochem Research) to adjust to a 0.25 mg / ml HSA solution. 0.2 ml was used for immunization. Before and 8 days after the salt immunization, blood was collected from the chimeric mice, and human antibodies / chains and human antibodies / c chains in the serum were detected using an ELISA method (see Example 14). 50mM carbonic acid-bicarbonate-61-This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) 1223585 A7 B7 V. Description of the invention (59) Anti-human antibody diluted in salt buffer pH9.6 / c Chain sheep antibodies were coated on 96-well microtiter plates, serum samples were added, followed by biotin-labeled anti-human antibody / c chain sheep antibodies (VECTOR LABORATORIES, INC., BA-3060), and then biotin was added. After the complex of peroxidase and antibiotics DH (VECTOR LABORATORIES, INC., Vectastain ABC kit PK4000) was cultivated, the peroxidase was used as a substrate, and 3,3 ^ 5,5, tetramethyl group was added. Aniline (TMBZ, Sumitomo Dolite, ML-1120T) was used to evaluate enzyme activity at 450 nm. Refined human IgG (Xigoma, 1-3 889) with a known / C chain and a known concentration was used as the standard, and PBS was added in a stepwise dilution. For #chain, anti-human // chain mouse single-source antibody (Xigoma, 1-63 85) diluted in 50 mM carbonate-bicarbonate buffer pH 9.6 was applied to a 96-well microtiter plate, and serum was added. The sample was then cultured with peroxidase-labeled anti-human # chain mouse antibody (The Binding Site Limited, MP008), and enzyme activity was evaluated by adding TMBZ (Sumitomo Berate, ML-1120T) with an absorbance of 450 nm. Purified human IgM (Oganon technology, 6001-1590) standard with a purified // chain and known concentration was diluted stepwise with PBS supplemented with mouse serum (Xigoma, M5905). As a result, both the human antibodies and the / chain were detected before the immunization in both individuals. The serum concentration increased after immunization (Tables 7 and 8). Table 7. Human antibody concentration in chimeric mouse KPG15 (ELISA)

IgM (mg / liter) Ig / c (mg / liter) Before Immunization 0.19 1.6 The 8th day after Immunization 0.75 1.7 -62- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm) Binding

Line 1223585 A7 B7 V. Description of the invention (60) Table 8. Human antibody concentration in chimeric mouse KPG18 (ELISA)

IgM (mg / litre) Ig / c (mg / litre) 0.29 0.57 before immunization 3.4 0.87 after immunization From this result, chimera was obtained from ES cells that retained human chromosome 2 fragment and 14 chromosome partial fragment In mice, the functions of human antibody heavy chain and light chain genes were confirmed. (Example 21) An anti-HSA human antibody τ chain was detected in the serum of a chimeric mouse introduced with a human chromosome 14 fragment. The chimeric mouse (K9) with a human chromosome 14 fragment prepared in the same manner as in Example 10 was retained. , K11; both from TT2 cell line 3-2, with chimeric rates of 50% and 30%, respectively, 4 times after 79 days, 93 days, 107 days, and 133 days (K9), or 74 days after birth , 88 days, 111 days (K11) three times, and HSA was immunized in the same manner as in (Example 20). The human 7-chain antibody against human serum albumin in the serum of this chimeric mouse was detected by ELISA (see Example 14). HSA (Xigoma, A3782) diluted in 50nm carbonate-bicarbonate buffer pH9.6 was applied to a 96-well microtiter plate, and a sample was added, followed by peroxidase-labeled anti-human IgG mouse antibody. (Invention, 08007E) After incubation, using peroxidase as the substrate, by adding o-phenylenediamine (OPD, Sumitomo Dolite, ML-1 1300), the enzyme activity was evaluated at an absorbance of 490 nm, and chimeric immunized with HSA The potency of anti-HSA human IgG in mouse serum increased after immunization. Antibodies after HSA immunization in control ICR mice -63- This paper size applies Chinese National Standard (CNS) A4 (210 X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (61) The strength of HSA human IgG is background level. The results are shown in (Fig. 17 and Fig. 17, the horizontal axis represents the number of days after HSA immunization in the chimeric mice, and the vertical axis represents the absorbance at 490 nm. As a result, the chimerism of the human chromosome 14 fragment was retained. In mice, it was confirmed that the antibody value of antigen-specific human IgG had increased in response to HS A antigen stimulation. (Example 22) Human antibody lambda bonds were detected in the serum of chimeric mouse introduced with human chromosome 22 fragment for 9 months Aged chimeric mice (Example 3, K22-7; chimerism rate 10%), blood was collected, human antibodies in serum; I chain, detected by ELISA method (refer to Example 14). Anti-human antibodies diluted in bicarbonate buffer pH 9.6; I-chain sheep antibodies (VECTOR LABORATORIES, INC, AI-3 070) coated 96-well microtiter plates, serum samples were added, followed by biotin-labeled human antibodies Lambda-chain sheep antibodies (VECTOR LABORATORIES, INC., BA-3070) cultured, plus biotinylated Amaranth peroxidase and a compound of antibiotics DH (VECTOR LABORATORIES, INC., Vectastain ABC kit PK4000) After incubation, using peroxidase as the substrate, by adding TM BZ (Sumitomo Berate, ML-1120T), the enzyme activity was evaluated by the absorbance at 450nm. The purified human IgG (Xigoma, 1-40 14) with a known lambda chain concentration was used as the standard, and mouse serum was added. The PBS was diluted stepwise. The antibody lambda chain corresponding to a concentration of human IgG of 180 nanograms / ml was detected in the chimeric mice. As a result, human chimeric mice retaining human chromosome 22 were identified as humans. The antibody lambda chain gene is functional. (Example 23) Chimeric mouse serum test for introduction of human chromosome 2 fragment -64- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) binding

Line 1223585 A7 B7 V. Explanation of the invention (62) Human antibody / C bond from 5-week-old chimeric mice (Example 13, K2-8, chimeric rate 70%) and 9-week-old chimeric mice Rats (Example 13, K2-3, K2 · 4, K2-12, and chimerism rates of 50%, 20%, and 80%, respectively) were collected, and human antibodies / c chains in serum were detected using ELISA (Example 14). An anti-human antibody / c-chain sheep antibody (VECTOR LABORATORIES, INC., AI-3060) diluted in 50 nm carbonate-bicarbonate buffer pH 9 · 6 (VECTOR LABORATORIES, INC., AI-3060) was applied to a 96-well microtiter plate, and a serum sample was added, followed by Add biotin-labeled anti-human antibody / c-chain sheep antibody (VECTOR LABORATORIES, INC ·, BA-3060) to culture, then add biotinylated camellia peroxidase and antibiotic DH complex (VECTOR LABORATORIES, INC ·, Vectastain ABC kit) After incubation, TMBZ (Sumitomo Berate, ML-1120T) was added to evaluate enzyme activity at 450 nm. The results were shown in Table 9 by using IgG (Xigoma, 1-3889) of purified IgG / c chain as a standard and PBS with mouse serum added in stages. Table 9. Human antibody / c chain concentration (ELISA) binding in chimeric mice

Ig / c (mg / l) of post-intraline mice K2-3 124 K2-4 85 K2-8 25 K2-12 56-Also, chimeric mice that retain human chromosome 2 fragments (Example 13, K2- 3, and K2-4), three times after the 66th, 80th, and 102nd days after birth, and (Implementation -65- This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) 1223585 A7 B7 _ 5 Explanation of the invention (63) Example 20) Similarly, HSA was immunized. The chimeric mouse (K2-12) was detected 4 times at 63, 77, 91, and 116 days after birth. The human antibody / c chain of HSA in the serum of the chimeric mouse was detected by ELISA ( Refer to Example 1. Sent HSA (Xigoma, A3782) diluted in 50 mM carbonate-bicarbonate buffer pH 9 · 6 to a 96-well microtiter plate, add a sample, and then add biotin-labeled antibody Human antibody / C-chain sheep antibody (VECTOR LABORATORIES, INC., BA-3060) culture, plus a complex of biotinylated mandala peroxidase and antibiotic DH (VECTOR LABORATORIES, INC., Vectastain ABC kit) After incubation, the enzyme activity was evaluated with absorbance at 490 nm using peroxidase as the substrate and the addition of OPD (Sumitomo Berate, ML-1 1300). Anti-HSA human / C in chimeric mouse serum immunized with HSA Chain valence increased after immunization. On the one hand, in control ICR mice, the valence of anti-HSA human antibody / c chain after HSA immunization was at the background level. The results are shown in (Figure 18). In Figure 18, horizontal The axis indicates the number of HSA in the chimeric mice after the initial immunization, and the axis indicates the absorbance at 490 nm. As a result, it was confirmed that the human antibody / C chain gene had a function in the chimeric mouse retaining the partial fragment of the human second chromosome, and that the antibody value of the antigen-specific human Ig / c increased in response to HSA antigen stimulation. (Example 24) A hybridoma producing a human antibody heavy chain (#chain or 7chain) was obtained from a chimeric mouse introduced into the human chromosome 14 from (embedded in Example 21) a chimeric mouse K9 immunized with HSA. The spleen was taken out and fused with myeloid tumor cells to make hybrid tumors. The method of making hybrid tumors was <Anto Chiba, Introduction to Single-source Antibody Experiments, Kodansha-66-This paper is in accordance with China National Standard (CNS) A4 (210X297 Mm)

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Line 1223585 A7 B7 V. Description of the invention (64) Science, 1991 &gt; As the method described, as a myeloid tumor cell, Sp-2 / 0-Agl4 (Daibenben Pharmaceutical, 05-554) was used. 10% of ORIGEN hybridoma colony selection factor (HCF, Polk water, Brown) was added to the culture solution, and it was spread on 8 96-well plates. After 3 days, G418 1 mg / ml was added to the culture solution. After 1 to 3 weeks of culture, the culture supernatant was analyzed by ELISA. A human #chain mouse single-source antibody (Xigoma, 1-63 85) diluted with 50 mM carbonate-bicarbonate buffer pH 9.6 was applied to a 96-well microtiter plate, and the sample diluted with PBS was added. Then add peroxidase-labeled anti-human #chain mouse antibody (The Binding Site Limited, MP008), and after culture, use 2,2-azinobis (3-ethyl p seroline-6-contanoic acid) as a substrate The diammonium salt (ABTS, Kirkegaard &amp; Perry Laboratories Inc., 04-10-17) was detected, and 7 positive holes were obtained from the chain. For the r chain, a single human anti-human T chain mouse antibody (Xigoma, I-6260) was coated on a 96-well microtiter plate, and a sample diluted with PBS was added, followed by peroxidase-labeled anti-human 7 After the chain mouse antibody (Invention, 08007E) was cultured, two human antibody r chain positive holes were detected with ABTS (Kirkegaard &amp; Perry Laboratories Inc., 04-10-17). (Example 25) A hybridoma producing a human antibody light chain was obtained from a chimeric mouse into which human chromosome 2 was introduced. (Example 23) Chimeric mouse K2-3 immunized with HSA was removed at 105th day after birth. Fusion with myeloma cell to make hybrid tumor. The method for producing hybridomas is based on &lt; Introduction of Anton Chiba, Single-source Antibody Experimental Operation, Social Sciences, 1991 &gt;. As a method for myeloid tumor cells, P3 X 63Ag8.653 (Okumoto Pharmaceutical, 05-565) is used. ). 10% HCF (Poker Water Brown) was added to the culture medium, and it was spread on 10 96-well plates. After 3 days, -67- This paper size applies to China National Standard (CNS) A4 (210X 297mm) binding

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G418 was added to the culture solution at 1 mg / ml, and cultured for 1 to 3 weeks. The pores in the colonies were cultured and the supernatant was cultured with £ 1 ^ § eight methods. The method was performed in the same manner as in (Example 23) to obtain the human antibody "chain-positive asexual breeding line." (Example 26) Re-labeling of puramycin resistance on the human chromosome 22 by G4 i 8 resistance-resistant gene mapping For the retention of eighty-nine cells of human chromosome 22 labeled with G418 resistance (_ 22: 2, shellfish (Obtained in Example 1), and relabeled with ptomycin resistance of human 4 chromosome 22 in the same manner as in Example 16). In ^ 2 cells, 200 pods of the dual-drug-resistant strain community obtained by pPGKPuro electroporation were used as a group, and 3 groups (η, p2 '㈠) were used as the donor cells. The wild-type mouse A9 cells were transplanted. As a result, 6 were obtained from P2, 3 were obtained from P2, and 3 were obtained from p3. The two agents were resistant to asexual reproduction. Among them, by using 6-1 from P3 as the microcell donor cells and wild type A9 cells as the sister cells, a microcell transfer experiment was performed to confirm that human chromosome 22 was additionally labeled with pramycin resistance gene (implemented (Example 18). The acquisition of microcells and the fusion with A9 cells were performed in the same manner as in (Example 丨). As a result, after the microcells moved into the next day, 28 G4 18 resistant communities appeared. For these communities, exchange 厶 8 After micrograms / ml of ploromycin medium, after culturing for 3 days, ^ (75 /.) Colonies survived. In the microcell method, because of 1 donor cell: In many cases, only 1 Few or a few chromosomes, so the tolerance genes are simultaneously high Entry means that the human chromosome 22 labeled with 18 tolerance was retained for 6_ 丨, and was also labeled with the pulomycin resistance gene. (Example 27) Human antibody weight was obtained from a hybridoma producing a human antibody heavy chain. 68- This paper is suitable for financial standards (CNS) A4 specification (㈣X297 public love)! 223585 A7 B7 V. Description of the invention (66) The cDNA and the determinable base sequence of the variable strand are obtained in (Example 15) Among hybridomas producing human antibody heavy chain (IgM), total RNA was obtained from H4B7 (HSA-specific) and H8F9 (non-specific) using ISOGEN. The synthesis of cDNA was performed using Ready-To-Go T -primed 1st strand kit (Pharmacia), using 5 micrograms of total RNA each. The resulting cDNA was obtained using primers shown below (see 1 ^ 1: 1 ^ 1 ^, etc., BIO / TECHNOLOGY, 7, 934 -, 1989, Word et al., Int. Immunol., 1,296-, 1989), PCR was performed to amplify the variable domain of human antibody heavy chain. CM1 (human IgM constant domain): 5, -TTGTATTTCCAGGAGAAAG TG (sequence number 45) CM2 (ibid.): 5, -GGAGACGAGGGGGAAAAGGG (sequence number 46) HS1 (human heavy chain variable Field): 5f-ATGGACTGGACCTGGAGG (AG) TC (CT) TCT (GT) C (Serial number 47) (mixture of 8 types) HS2 (ibid.): 5 *-ATGGAG (CT) TTGGGCTGA (GC) CTGG (GC) TT T (CT) T (Sequence No. 48) (mixture of 16 types) HS3 (ibid.): 5,-ATG (AG) A (AC) (AC) (AT) ACT (GT) TG (GT) ( AT) (GCT) C (AT) (CT) (GC) CT (CT) CTG (Sequence 歹 J # 49) (6144 mixtures) _

* () Indicates that the base at its position is a mixture made of any of (). H4B7, H8F9 are the first PCR, combining HS1 X CM1, HS2X -69- This paper size applies to China National Standard (CNS) A4 (210X297 mm) binding

Line 1223585 A7 ____B7_ V. Explanation of the invention (67) CM1, HS3X CM1 3 types of primers (94 ° C, 1 minute, 50 ° C, 2 minutes, 72 ° C, 3 minutes, 40 cycles, using Barkin Weimar Company, Shama Circulator) -140, re-amplify the PCR products with primers of HS1 X CM2, HS2 X CM2, and HS3X CM2, respectively (temperature conditions are the same as the previous description, 30 cycles), and the amplified products are After 1.5% agarose electrophoresis, it was detected by ethidium bromide staining. As a result, an extension product of about 490 bp was confirmed with the primer of HS3 XCM2 for H4B7. For H8F9, a weak loop was identified at the same position with the HS3 XCM2 primer, and the primer was re-amplified (temperature conditions are the same as the previous note, 30 cycles). As a result, the amplified product is detected very strongly as a signal. These PCR products were cloned from the Smal part of the pBlueScriptll SK + (Stratagene) vector according to the method of Ishida et al. (Genetic expression experiment operation, Kodansha science class, 1995). Inserted into the plastid of the amplification product. For # 2, # 3, # 4 (H4B7), # 11, # 13, # 14 (H8F9), the automatic fluorescence scanner (Applied Bio System) was used to determine the amplification. The base sequence of the product. Human antibody VH domain (Marks et al., Eur · J. Immunol. 21, 985-, 1991), JH domain (Ravetch et al., Cell, 27, 5 83) with reported base sequences and predicted amino acid sequences -, 1981). As a result of sequence comparison, it is understood that H4B7 and H8F9 are all formed by the combination of VH4 family and JH2. As a result, chimeric mice retaining partial fragments of human chromosome 14 have been shown to produce fully functional human anti-heavy chain proteins. (Example 28) Human antibody / c chain cDNA was obtained from the spleen of a chimeric mouse expressing a human antibody / c chain_Determining the base sequence of human antibody / c chain__ Obtained from (Example 13) and confirmed to express a human antibody in (Example 23) The spleen of the chimeric mouse K2-8 of the / c chain was combined with -70-feet in the same manner as in (Example 5); it was bound with the Chinese national ft (CNS) A4 size (210X297 mm)

Line 1223585 A7 B7 V. The cDNA of the invention description (68) 'Borrowed from the primers below (refer to Larrick et al., BIO / TECHNOLOGY, 7, 934-, 1989, Whitehurst et al., Nucleic Acids Res ·, 20, 4929-, 1992) ) Perform PCR to amplify human / c-chain variable domains. As a negative control group, cDNA derived from the liver obtained from K2-8 and spleen derived from the chimeric mouse K3-2-2 from TT2 / # 14 3-2 (Example 10) were used. KC2 (human Ig / C chain constant field): 5,-CAGAGGCAGTTCCAGATTT C (sequence number 50) KC3 (ibid.): 5, -TGGGATAGAAGTTATTCAGC (sequence ij number 51) KVMIX (human Ig / c chain variable field): 5f -ATGGACATG (AG) (AG) (AG) (AG) (AGT) (CT) CC (ACT) (ACG) G (CT) (GT) CA (CG) CTT (Order number 丨 J number 52) (mixture of 3456 kinds) * () Indicates that the base in its position is made by any one of (). The PCR was performed using a combination of KVMIXXKC2 and KVMIXXKC3 primers at 55 ° C for 15 seconds, 72 ° C for 20 seconds, and 40 cycles (using Bajin Weima Co., Thermal Cycler-9600). The amplified products were electrophoresed on 1.5% agarose and detected by ethidium bromide staining. As a result, the combination of the two detected the expected amplification products of about 420 bps (KC2) and about 450 bps (KC3) in length. On the one hand, no specific amplification products were detected in the two negative control groups. These amplified products were cloned from the Smal or EcoRV site of pBlueScdptll SK + (Stratagene) vector according to the methods of Ishida et al. Inserted into the plastid of the amplified product, there is one VK- # 1 asexual breeding line from KVMIXXKC2, with automatic fluorescence-71-This paper size applies Chinese National Standard (CNS) A4 (210X 297 mm) m binding

Line 1223585 A7 B7 V. Description of the invention (69) The scanner (Applied Bio System) determines the sequence of the motif of the amplified product. Since the obtained base sequence does not contain a stop codon from the start codon of the Ig / c chain to the constant domain, it is considered that the selected amplification product encodes a functional human Ig / c chain variable domain. In addition, the results of comparison with the base sequences of the reported human antibody V / C domain (Klein et al., Eur · J. Immunol ·, 23, 3248-, 1993) and the J / c domain (Whitehurst et al., Preface) show that In chimeric mice that retain a partial fragment of human chromosome 2, a fully functional human antibody / c-chain protein is produced. (Example 29) Detection and quantification of human antibody 7-chain subclasses and #chains in the serum of chimeric mice holding human chromosome 14 fragments from chimeric mice 11 weeks of age after (Example 10) (K15 A and K16A; from 1-4, chimerism rate 70, 50%) Blood was collected, and the concentration of human antibody r chain subclasses in the serum was detected according to (Example 14) using ELISA. [Measurement of human IgG1] Anti-human IgG antibody (Xigoma, 1-6260) was diluted with PBS, and coated on a 96-well microtiter plate. Serum samples were added, followed by peroxidase-labeled anti-human IgG1 antibody (Invention, 08027E) and cultured. Enzyme activity was evaluated at 450 nm by adding TMBZ (Sumitomo Berate, ML-1120T). The purified human IgG1 (Xigoma, 1-3889) at a known concentration was used as a standard, and mouse serum was added to the PBS for stepwise dilution. [Measurement of human IgG] Anti-human IgG2 antibody (Xigoma, 1-95 13) was diluted with PBS, and coated on 96-well microtiter plates. Serum samples were added, followed by incubation with peroxidase-labeled anti-human IgG antibody (Xigoma, A-0 170). Enzyme activity was evaluated at 450 nm with the addition of TMBZ (Sumitomo Berate, ML-1120T). Human IgG2 (Xi-72-) with a known concentration in this paper is applicable to the Chinese National Standard (CNS) A4 (210X 297 mm). Binding

Line 1223585 A7 B7 V. Description of the invention (70) Goma, 1-4139) is used as a standard, and the PBS is diluted stepwise with the addition of mouse serum. [Measurement of human IgG3] The anti-human IgG3 antibody (Xigoma, 1-7260) was diluted with 100 mM glycine acid buffer pH 2.5, left at room temperature for 5 minutes, and diluted with 100 mM phosphate buffer pH 7.0 10 Times, coated on a 96-well microtiter plate. Serum samples were added, followed by peroxidase-labeled anti-human IgG antibody (Invention, 08007E), and TMBZ (Sumitomo Berate, ML-1120T) was added to evaluate enzyme activity at 450 nm. Refined human IgG3 (Xigoma, 1-4389) was used as a standard, and mouse serum was added to PBS for stepwise dilution. [Measurement of human 1§04] The anti-human 400 antibody (Xigoma, 1-763 5) was diluted with 100 mM glycine hydrochloride buffer pH 2.5, and left at room temperature, and then buffered with 100 mM phosphate The solution was diluted 10-fold at pH 7.0 and applied to a 96-well microtiter plate. Serum samples were added, followed by peroxidase-labeled anti-human IgG antibody (Invention, 08007E). After incubation, TMBZ (Sumitomo Beitel, ML-1120T) was added to evaluate enzyme activity at 450 nm. The purified human IgG4 (Xigoma, 1-4639) at a known concentration was used as a standard, and mouse serum was added to PBS for stepwise dilution. [Measurement of human IgM] For // chain, anti-human / Z chain mouse single-source antibody (Xigoma, 1-6385) diluted in PBS was coated on a 96-well microtiter plate, and serum samples were added, followed by After the peroxidase-labeled anti-human #chain mouse antibody (The Binding Site Limited, MP008) was cultured, TMBZ (Sumitomo Beitel, ML-1120T) was added with peroxidase as the substrate, and the absorbance was evaluated at 450 nm Enzyme activity, refined with #chain concentration -73- This paper size applies Chinese National Standard (CNS) A4 (210X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (71) Known human IgM (Organon. Technology, 6001-1 590) as the standard, with PBS supplemented with mouse serum (Xigoma, M5905), stepwise diluted. The results are shown in Table 10. In chimeric mice K1 5 A and K16A, all IgG1, IgG2, IgG3, and IgG4 subtypes and IgM were detected. Table 10 Human antibody IgG subclasses and IgM concentrations (ELISA) in chimeric mice IgG1 IgG2 IgG3 IgG4 IgM (mg / l) K15A 2.25 1.96 0.17 0.43 7.09 K16A 0.30 0.69 0.10 0.07 0.87 (Example 30 ) Obtaining of mouse ES cell line (TT2) retaining human chromosome 22 To obtain mouse ES cell (TT2) retaining human chromosome 22, use 6.1 obtained from (Example 26) (A9 / # 22, G418, pramycin resistance) cell line. As a chromosomal receptor cell, a wild-type TT2 cell line was used (Example 9). The microcell fusion experiment and the selection of the promycin-resistant strains were performed at a concentration of 0.75 microgram / ml of the promycin, and the rest were performed in the same manner as in the case of selecting the G4 18-resistant strain of (Example 9). As a result, the appearance frequency of the protomycin-resistant strains was 2 per 107 TT2 cells, and cryopreservation of pramycin and genomic DNA acquisition were performed in the same manner as in (Example 2). Preservation of human chromosome 22 For pramycin-resistant strain PG22-1, the following (1) and (2) were confirmed. (1) PCR analysis using the genomic DNA of the puramycin-resistant strain as a template exists in the human 22 -74- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

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1223585 A7 B7 V. Description of the invention (72) Among the genes on the chromosome (Genetic Maps, preface), PCR amplification was performed on 10 primers confirmed to exist in (Example 2; A9 / # 22). All marks present in (Example 2; A9 / # 22). (2) Analysis of the southern aspiration method, using the human L1 sequence as a probe according to the method shown in (Example 2), for wild type TT2 from the negative control group, chromosome donor cells 6-1, and pramycin resistance TT2 Genomic DNA of cell line PG22-1 was performed. The results and results are shown in (Figure 19). The molecular weight of DNA is shown on the left in the figure. The pattern of the PG22-1's ring zone was the same as that of 6-1, and the signal intensity was also the same. Therefore, it was confirmed that the 22nd chromosome in the 6-1 cell line moved into PG22-1. From the above experiments, it was confirmed that the Pullo-resistant TT2 cell line PG22-1 retained all or most of the human chromosome 22. (Example 31) Mouse ES cell line (TT2) that retains human chromosome 22 was produced. Chimeric mice were obtained from frozen stock (Example 30) and confirmed to retain human chromosome 22 pulomycin. Eight-cell stage embryos obtained by mating ICR or MCH (ICI1) (Kelia, Japan) male and female mice were injected with 10 to 12 embryos. The cells were cultured overnight in ES cell culture medium (Example 9), and after placental cells occurred, the uterus of parental ICR mice (Japan Crelia) was temporarily borrowed 2.5 days after pseudopregnancy treatment. 10 injection embryos. The results of mouse production are shown (Table 11). The results of 266 implanted embryos were transferred and 36 daughter mice were born. Chimeric individuals can be judged by identifying whether the coat color is white from the host embryo (ICR) with a wild color (strong tea) from TT2 cells. Of the 36 births, the hair color obviously has a wild color, that is, can be -75- This paper size applies to China National Standard (CNS) A4 (210X297 mm) binding

Line 1223585 A7 B7 V. Description of Invention (73) There are 8 individuals who have identified the contribution of ES cells. From this result, it was confirmed that the ES cell line (derived from TT2, PG22-1) retaining the human chromosome 22 was provided with chimeric forming ability, that is, the ability to differentiate into normal tissues of mouse individuals. Table 11. Chimeric mouse prepared from TT2 cell line retaining human chromosome 22 -76- This paper is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) 1223585 A7 B7 V. Description of the invention (74 ES cell line / Contributing rate of puplomycin to ES cell-injected mice Sui Hexiao to coat color Human chromosome tolerance strain No. 8 Cell stage embryo number Number of rats ~ 20% 20-50% 50-80% TT2 / # 22 PG22- 1 266 36 4 1 3

(Example 32) Detection and quantification of human antibody lambda chains in the serum of chimeric mice retaining human chromosome 22

The concentration of human antibodies in the serum of the chimeric mouse KPG22-1 to 3 (Example 31) was quantified according to (Example 14) using the ELISA method. Blood was collected from chimeric mice 2 months after birth, and human anti-λ chains were detected by ELISA. Anti-human immunoglobulin and chain antibody (VECTOR LABORATORIES, INC., IA-3070) diluted in PBS was coated on a 96-well microtiter plate, a serum sample was added, and then a biotin-labeled anti-human immunoglobulin was added Chain antibody (VECTOR LABORATORIES, INC., BA-3070) was cultured, and then a complex of biotinylated mandala peroxidase and antibiotic DH (VECTOR LABORATORIES, INC., Vectastain ABC set) was cultured, and then added by TMBZ (Sumitomo Berate, ML-1120T), the enzyme activity was evaluated with an absorbance of 450nm, the refined human IgM (Oyster Pharmaceuticals, U13200) with a lambda chain and a known concentration was used as the standard, and mouse serum was added to PBS Staged dilution. The results are shown in Table 12. From these results, it was confirmed that the chimeric mouse retaining the chromosome 22 has the function of the human antibody λ chain gene. -77- The size of this paper applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1223585 A7 B7 V. Description of invention (75) Table 12. Human antibodies in chimeric mice; I chain concentration (ELISA) post-chimeric mouse chimeric rate% Ig A (mg / L) KPG22-1 50 12 KPG22-2 50 18 KPG22-3 20 24 (Example 33) Anti-human HS A humans were detected in the serum of chimeric mice introduced with human chromosome 22; chimeric mice of I chain (Example 31, KPG22-3) were born on the 79th, 94th, and 10th day after birth Next, HSA was immunized in the same manner as in (Example 20). Human antibodies in serum; I chain was detected by ELISA (Example 14). Dilute 50 mg of carbonic acid-bicarbonate buffer ρΗ9 · 6 to 7 g of HSA (Xigoma, A3782) in a 96-well microtiter plate, add serum samples, and then add biotin-labeled anti-human Immunoglobulin lambda chain antibody (VECTOR LABORATORIES, INC ·, BA-3070) cultured, followed by biotinylated camellia peroxidase and antibiotic DH complex (VECTOR LABORATORIES, INC ·, Vectastain ABC kit) culture , By adding TMBZ (Sumitomo Berate, ML-1120T), the enzyme activity was evaluated with an absorbance of 450 nm. The anti-HS A human λ chain potency in the serum of chimeric mice immunized with HS A increased after immunization. On the one hand, in ICR mice as a control group, the power of the anti-HSA human antibody lambda chain after HSA immunization is background. The results are shown in (Figure 20). In Fig. 20, the horizontal axis indicates the number of days when the chimeric rabbit was first immunized with HSA, and the vertical axis indicates the absorbance at 450 nm. -78- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) binding

Line 1223585 A7 B7_____ V. Description of the invention (76) Based on these results, it was confirmed that in the chimeric mouse retaining human chromosome 22, human antibodies have chain genes functioning. In addition, for HSA antigen stimulation, antigen-specific human Ig λ occurred. The antibody price rose. (Example 34) A hybridoma producing a human antibody light chain was obtained from a chimeric mouse into which human chromosome 22 was introduced. In the same manner as in (Example 25), a chimeric mouse immunized with human albumin was used in (Example 33). KPG22-3, the spleen was taken out on the 113th day after birth, and it was fused with myeloma cell to make a hybrid tumor. The method for producing hybridomas is based on the method described in "Anton Chiba, Introduction to Single-source Antibody Experiments, Kodan Social Science, 1991", and SP-2 / 0-Agl4 (Oyoshimoto Pharmaceutical Co., Ltd.) is used as myeloma tumor cells. 05-554). 10% HCF (Weir Brown) was added to the culture medium and spread on five 96-well plates, cultured for 1 to 3 weeks, and the culture supernatants showing the holes in the colonies were analyzed by ELISA. ELISA method and (Example 33) The same method is used to obtain asexual breeding lines that are positive for human antibody Λ bond. (Example 35) Obtaining a mouse ES cell line that simultaneously retains a partial fragment of human chromosome 22 and a partial fragment of chromosome 14 To obtain mouse ES cells that simultaneously retain a partial fragment of human chromosome 22 and 14 With cells, 6-1 (A9 / # 22, G41 8, pramycin resistance) cell line obtained in (Example 26) was used. As a chromosomal receptor cell, a Q4i8-resistant TT2 cell line 1 to 4 in which a partial fragment of the human chromosome 14 has been retained (Example 9). The microcell fusion experiment and the selection of a promycin-resistant strain were performed at a concentration of 0.775 micrograms / ml of promycin, and the rest were performed in the same manner as in the case of selecting the G418-resistant strain of (Example 9). The results show that the protomycin-resistant strains appear at a frequency of 1 cell per 1 2 -79-

1223585 A7 B7 V. Description of invention (77). These prosomycin-resistant strains also proliferated in the presence of 300 micrograms / ml of G418, so it was confirmed that G4 18 tolerance was also maintained. Cryopreservation of double-drug resistant strains and genomic DNA acquisition were performed in the same manner as in (Example 2). The retention of human chromosome 22 and partial fragments of human chromosome 14 was confirmed by the following PCR analysis of the dual drug-resistant strain PG22-5. Based on the genomic DNA of the dual-drug-resistant strain as a template, it is present in genes (Genetic Maps, preface) on human chromosomes 22 and 14. For chromosome 22, it is confirmed that it exists as (Example Saint; A9 / # 22). Chromosome 14 confirmed the results of PCR amplification with each primer present in (Example 9; TT2 / # 14 1-4). For chromosome 22, there were 3 markers (D22S275, D22S315, IgA), For chromosome 14, all markers present in TT2 / # 14 1-4 were detected. From the above experiments, the obtained double-resistant TT2 cell line can confirm that the human chromosome 22 partial fragment and the 14 chromosome partial fragment are retained at the same time. (Example 36) A mouse ES cell line containing both the human chromosome 22 fragment and the 14 chromosome partial fragment was made. Chimeric mice were obtained from the frozen stock (Example 35), and it was confirmed that the human 22nd fragment was retained. Chromosome partial fragment and 14th chromosome partial fragment G4 18, pulomycin dual-resistant TT2 cell line PG22-5, obtained by mating male and female mice of ICR or MCH (ICR) (Benkeria) In 8-cell stage embryos, 10 to 12 embryos are injected. The cells were cultured overnight in ES cell culture medium (Example 9), so that after placental cells occurred, the uterus of parental ICR mice (Nihon Kelia) was temporarily borrowed 2.5 days after the pregnancy treatment. About 10 embryos were injected. -80- This paper size applies to China National Standard (CNS) Α4 size (210X 297mm) binding

Line 1223585 A7 B7 V. Description of the invention (78) The results of the mosaic production are shown in (Table 13). The result of transplantation was 302 embryos, and 16 daughter mice were born. The chimeric individuals were judged by whether the coat color was confirmed to have a wild color (strong tea) from TT2 cells in the white from the host embryo (ICR). It is said that among the 16 animals, the hair color obviously has a wild color part, that is to say, there are 5 individuals who are considered to have contributed by ES cells. From this result, the ES cell line (PG22-5) retaining the human chromosome 22 fragment and the 14th chromosome fragment was confirmed to retain the chimeric forming ability, that is, the ability to differentiate into normal tissues of mouse individuals. Table 13 Chimeric Mouse ES Cell Lines Made from TT2 Cells Retaining Human Chromosomes 22 and 14 / Dual Tolerance Injected ES Cells Mouse Chimeric Contribution to Hair Color Number of Human Chromosome Numbers Count ~ 20% 20-50% 50-80% _8 cell stage embryos _ TT2 / # 22 + # 14 PG22-5 302 16 5 3 2 0 (Example 37) From the same time while retaining the human chromosome 22 fragment 14th Detection and quantification of human antibody λ chain and human antibody V chain in the serum of chimeric mice of ES cells of partial chromosomes. For chimeric mice (KPG22-9, 10, and 12) of Example 36, HSA immunity. KPG22-9 and KPG22-10 were immunized at 11 weeks of age, and blood was collected 2 weeks later. KPG22-12 was immunized at 7 and 9 weeks of age, and blood was collected 2 weeks after the second immunization. Serum has human antibody // chain and human antibody λ chain, and another human -81-This paper size applies Chinese National Standard (CNS) Α4 specification (210 X 297 mm)

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Line 1223585 A7 B7 V. Description of the invention (79) Antibodies of λ chain and human μ chain were detected by ELISA method according to (Example 14). For detection of fully human antibodies, anti-human immunoglobulin lambda chain antibody (Kirkegaard &amp; Perry Laboratories Inc., 01-10-11) diluted in PBS was coated on 96-well microtiter plates, and serum samples were added. Next, add peroxidase-labeled anti-human immunoglobulin #chain antibody (The Binding Site Limited, MP008), and then use peroxidase as the substrate and add 450MB with TMBZ (Sumitomo Berate, ML-1120T). The absorbance was used to evaluate the enzyme activity, and compared with human IgM (Dayokumoto Pharmaceutical Co., Ltd. U13200) (reduced stepwise with PBS supplemented with mouse serum), which was purified, with I chain and known concentration, to determine the concentration of human antibody in the serum. Human antibodies // chains and human antibodies The λ chain was detected and quantified in the same manner as in (Example 29) and (Example 32) by ELISA. The results are shown in Table 14. In chimeric mice, both the lambda chain and // chain were detected. Antibodies with # and λ chains of human antibodies were also detected. As a result, chimeric mice derived from ES cells retaining partial fragments of human chromosome 22 and partial fragments of chromosome 14 have the function of both the human antibody lambda chain gene and the human antibody # chain gene. In some cells, the heavy chain Both the light chain and the light chain were confirmed to produce complete human antibodies. As a control, the sera of the chimeric mouse (K9) retaining only human chromosome 14 (Example 10) and the chimeric mouse (KPG22-2) retaining only human chromosome 22 (Example 31) were determined. The concentrations of antibodies with human antibodies; I chain and # chain are background levels, and the detection here confirms that only complete antibodies with human λ chain and # chain are detected. -82- This paper size applies to China National Standard (CNS) Α4 size (210X297mm) binding

Line 1223585 A7 B7 V. Explanation of the invention (80) Table 14 Human antibody concentration in chimeric mice (ELISA) ES clonal line chimeric mice Chimeric rate% IgM (mg / L) Ig λ IgM, λ (mg / L) (mg / L) PG22-5 KPG22-9 30 2.54 9.9 0.043 PG22-5 KPG22-10 5 4.96 21.5 0.333 PG22-5 KPG22-12 40 3.71 7.0 0.048 3-2 K9 50 6.66-&lt; 0.003 PG22-1 KPG22-2 50-17.6 &lt; 0.003 (Example 38) Human antibodies were detected in chimeric mouse sera from ES cells that retained both human chromosome 2 fragment and 14 chromosome fragment / C chain and human #chain antibody) For the chimeric mouse KPG-15 (derived from the TT2ES asexual breeding line PG5, the chimeric rate was 10%) prepared in (Example 19), 2 to 3 months of age after birth In the meantime, human serum albumin (HSA, Xi Goma, A3782) dissolved in PBS and adjuvant (MPL + TDM emulsion, RIBI Immunochem Reseach) were mixed and adjusted to 0.25 mg / ml of HSA; immunized 3 times with 0.2 ml , Blood collection. Blood was collected from a chimeric mouse KPG-26 (derived from the TT2ES clone PG6 with a chimerism rate of 40%) at 6 weeks of age (Example 19). The complete human antibody concentration in the serum was detected by ELISA method according to (Example 14). Anti-human immunoglobulin / C-chain antibody (Kirkegaard &amp; Perry Laboratories, 01-10-10) diluted in PBS was coated on a 96-well microtiter plate and added with mouse serum (Xigoma, M5905) PB S diluted serum sample -83- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (81), and then add peroxidase-labeled anti-human immunoglobulin // chain antibody (The Binding Site Limited, MP008), and then use peroxidase as the substrate and add TMBZ (Sumitomo Berate, ML-1120T), the enzyme activity was evaluated at 450nm absorbance, and human IgM (Organon technology, 600 1-1 590) with refined / c chain and known concentration was used as the standard to add mice The serum was diluted PBS stepwise. The concentration of the / C chain and / or chain was determined in the same manner as in (Example 20). The results are shown in Table 15. Antibodies with human antibodies // bonds and / c chains were detected. In the serum of the chimeric mouse (K9) retaining only human chromosome 14 (Example 10) and the chimeric mouse (K2-9) retaining only human chromosome 2 (Example 13) For human antibodies, the concentration of the antibody with / c chain and / or chain is 0.002 mg / ml or less as the background level. From this result, it was confirmed that the human antibody / c chain gene and the human antibody // chain gene function simultaneously in the chimeric mouse derived from the ES cells retaining the human chromosome 2 fragment and the 14 chromosome partial fragment. Cells, heavy chains and light chains all produce fully human antibodies. Table 15 Human antibody concentrations in chimeric mice (ELISA) ES asexual reproduction hinders the chimeric rate of mice% IgM (mg / L) Ig / c IgM, / c (mg / L) (mg / L ) PG-5 KPG15 10 0.18 1.01 0.075 PG-6 KPG26 40 1.52 1.26 0.018 3-2 K9 50 6.66-below 0.002 5-1 K2-9 40-135 below 0.002 -84- This paper size applies to the Chinese National Standard (CNS) A4 size (210 X 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (82) (Example 39) Obtaining a mouse ES cell line (TT2F, X0) that retains a partial fragment of human chromosome 2 (X0) was used as a chromosome donor cell in the PG1 cell line obtained in (Example 16). As a chromosome receptor cell, it used the chromosome composition of (39, X0) to efficiently differentiate into egg cells in chimeric mice (Xian Han Shinichi, Biological Manipulation Series 8, Gene Target, Yangtusha, 1995) Cell lines (purchased by Oriental Life Technologies). The microcell fusion experiment and the selection of the promycin-resistant strain were performed at a concentration of 0.75 microgram / ml of promycin, and the rest were performed in the same manner as in the case of selecting the G41 8-resistant strain of (Example 9). The results showed that the promycin-resistant strains appeared at a frequency of 5 out of 107 TT2F cells. Cryopreservation and genomic DNA collection of these promomycin-resistant strains were performed in the same manner as in (Example 2). The retention of partial fragments of human chromosome 2 was analyzed for the drug-resistant strains P-20 and P-21 by the following PCR. Using the genomic DNA of the drug-resistant strain as a template, the gene (Genetic Maps, preamble) existing on the second chromosome, the primers C / c, FABP1, and the presence of (Example 1; A9 / # 2 w23) confirmed As a result of PCR amplification of three types of V / c 1-2, both strains confirmed the expected amplification products for all three types of primers. From the above experiments, the obtained promycin-resistant ES cell line (TT2F, XO) was confirmed to retain a portion of the human second chromosome. (Example 40) A chimeric mouse was produced from a mouse ES cell line (TT2F, XO) that retains a partial fragment of the human second chromosome. It was obtained from (Example 39) from the frozen stock, and it has been confirmed that the human -85 -This paper size applies to China National Standard (CNS) A4 (210X297mm) binding

Line 1223585 A7 B7 V. Description of the invention (83) 2 Ploomycin-resistant TT2F cell line P-21, a partial fragment of chromosome 2, was obtained by mating male and female mice with ICR or MCH (ICR) (Kelia Japan) In 8-cell stage embryos, 10 to 12 embryos are injected. 2. Culture overnight in ES cell culture medium (Example 9). After placental cells occur, 2.5 days after pseudopregnancy treatment, the uterus of the parental ICR mouse (Japan Klia) is transplanted in each unilateral uterus. About 10 embryos were injected. As a result of implantation of 141 embryos, 20 daughter mice were born. Chimeric individuals were judged by the color of their hairs, which confirmed whether there was a wild color (strong tea) from TT2 cells in the white color from the host embryo (ICR). The results of the mosaic production are shown in (Table 16). Of the 20 births, the hair color obviously has a wild color part, which means that there are 9 individuals who can be identified as contributing by ES cells. Four of them were chimeric mice from (ES cells) with completely wild hair. From this result, it was confirmed that the ES cell line (P-21) which retains a partial fragment of the human chromosome 2 retains the chimerism forming ability, that is, the ability to differentiate into normal tissues of mouse individuals. Table 16 Preparation of Chimeric Mouse ES Cell Lines from TT2F Cell Lines Retaining Partial Fragments of Human Chromosome 2 Contributing Rate of Chimeric Mice Injected into ES Spleen Mouse Chimera to Hair Color / 8th Number of rats ~ 20% 20-50% 50-80% 100% Chromosomal strain number Cell stage embryo number TT2F / # 2fg. P-21 141 20 9 0 2 3 4 (Example 41) From the second human reserve Chromosome part of the TT2F embedded paper size is applicable to China National Standard (CNS) A4 (210X297 mm) binding

-86- 1223585 A7 B7 V. Description of the invention (84) Detection and quantification of human antibody / C chain in the serum of mice (from Example 40) Chimeric mice (from Example 1) P-21, chimerism rate 100%, K2-1F ~ 4F) Blood was collected, and human antibody / C chain concentration in serum was quantified by ELISA in the same manner as in Example 20). The results are shown in Table 17. The use of ES cells as TT2F also confirmed the potential of human antibody / C chain genes in chimeric mice. Table 17 Human antibody / C chain concentration in chimeric mice (ELISA) Chimeric mouse chimeric rate% IgW mg / L K2-1F 100 66 K2-2F 100 156 K2-3F 100 99 K2-4F 100 20 (Example 42) The offspring of a chimeric mouse derived from mouse ES cells (TT2F, XO) that retain a partial fragment of the human second chromosome were detected. In mice, we examined whether K2-1F and K2-4F (100% chimeric rate of hair pieces) were mated with male ICR mice to produce offspring from ES cells. In this mating, among chimeric mice fertilized by sperm from ICR male mice (coating, inferiority), eggs from TT2F cells (wild color: dominant) give birth to wild colors, and eggs from ICR give birth to white children . All surviving child mice (K2-1F: 10, K2-4F: 5) obtained through one-time mating are all shown as derived from ES cells-87- This paper applies Chinese National Standard (CNS) A4 Specifications (210 X 297 mm)

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Line 1223585 A7 B7 V. The wild color of the hair color of the invention description (85). For genomic DNA modulated from the tails of these children, the retention of human chromosome fragments was examined by PCR. For the results of PCR amplification of three primers confirmed in P-21 (Example 39), SP-21 was confirmed in 4 of 10 (K2-1F) and 5 of 2 (K2-4F). The existence of the three kinds of marks. The PCR results of 15 daughter mice are shown (Figure 21). In the figure on the right, the molecular weight of the DNA (PX174, Haelll fragment, Japanese) and the main loop bands are shown. On the right, DNA from the tails of the parental chimeric K2-1F and K2-4F as the positive control group is used. These results show that the TT2F cell line P-21, which retains a partial fragment of the human second chromosome, differentiates into a functional egg in the chimeric mouse, and the partial descendant of the human second chromosome is transmitted from the offspring of the egg. (Example 43) Chimeric mouse offspring derived from mouse ES cells (TT2, XY) retaining partial fragments of human second chromosome were detected and the human chromosomes were retained in the chimeric mouse obtained in (Example 13), K2-1 8 (male, chimerism rate of 70%) was mixed with K2-19 (female, chimerism rate of 60%) and non-chimeric females with the same abdomen to examine whether offspring were born. TT2 cells have a chromosome structure of (40, XY), so the chimeric chimeric K2-1 8 has the potential to differentiate into functional sperm. In that case, wild-colored mice were born from eggs from ICR (white: disadvantaged) fertilized by sperm from TT2 cells (wild-colored, dominant) in the chimeric mouse. Out of 110 viable child mice obtained by mating, 10 showed coat color from ES cells. For genomic DNA prepared from the tails of 7 of 10 wild-colored mice, the retention of human chromosome fragments was examined by PCR. For the two types of primers C / c, FABP1 confirmed to be present in 5-1 strain (TT2 / # 2fg., Example 12) and -88- shown in (Example 1), this paper size applies the Chinese national standard ( CNS) A4 size (210X 297mm) binding

Line 1223585 A7 B7 V. Description of the invention (86) The results of PCR amplification of the V / c 1-2 primers confirmed that the presence of all three kinds of markers was confirmed in two of the seven. This result indicates that the TT2 cell line which retains a partial fragment of the human chromosome 2 differentiates into functional sperm in the chimeric mouse, and expresses that the partial fragment of the human chromosome 2 is transmitted in the descendants of the sperm. (Example 44) Detection and quantification of human antibodies / c chains in the serum of chimeric mouse offspring (Example 42) Chimeric mouse offspring K2-1F-1 to 10 and K2-4F-1 to 5 The human antibody / c chain concentration in serum was quantified by ELISA method. Blood was collected from mice about 4 to 6 weeks old after birth, and the human antibody / c chain in the serum was detected by the ELISA method in the same manner as in (Example 20). The results are shown in Table 18 together with the results of chromosome retention obtained in Example 42. The offspring born from the chimeric mice also confirmed that the human antibody / c chain gene functions. -89- This paper size applies Chinese National Standard (CNS) A4 (210X 297 mm) 1223585 A7 B7 V. Description of the invention (87) Table 18 Human antibody / C chain concentration (ELISA) in chimeric mouse offspring Parental chimeric mouse chimeric individual numbering Human chromosome fragment Ig / c (mg / l) K2-1F # 1-0.58 K2-1F # 2 + 84.1 K2-1F # 3 + 12.8 K2-1F # 4 + 15.1 K2-1F # 5-0.52 K2-1F # 6-0.58 K2-1F # 7-1.30 K2-1F # 8-0.90 K2-1F # 9-0.56 K2-1F # 10 + 28.8 K2-4F # 1-0.04 K2-4F # 2 + 23.3 K2-4F # 3 + 11.8 K2-4F # 4-0.08 K2-4F # 5-0.06 (Example 45) Analysis of chimeric mouse spleen cells introduced into the human chromosome 14 fragment Through the analysis of flow cell photometry, the method described in the following documents is used. 12 Biochemistry I-Immune Cells, edited by the Benson Biochemical Society, and lectures on freshman chemistry experiments. Cell Caine-1989, Tokyo Chemical Associates; University of Tokyo Medical-90-This paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 mm) 1223585 A7 B7 V. Description of the invention (88) Cancer Research Institute of Science Compiled by the Research Department, Cell Engineering Special Volume 8 New Cell Engineering Experiment Plan, 1991, Xiu Runsha; A. Doyle and JB Griffiths, "Cell and Tissue Culture: Laboratory Methods" (Cell &amp; Tissue Culture: Laboratory Procedures), published by John Wiley &amp; Sons Ltd., 1996. The spleen was removed from 6-month-old chimeric mice (KPG06; from PG16, chimerism rate 30%) after (Example 19), and PBS containing 1% of rat serum treated with aqueous ammonium chloride solution was added to PBS. In the experiment, fluorescein isocyanate (FITC) -labeled anti-mouse CD45R (B220) antibody (Invention, 01124A) was used to stain the cells. After washing, react with biotin-labeled anti-human IgM antibody (Invention Jian, 08072D) in PBS containing 5% mouse serum or as a control to react with biotin-labeled anti-human Ig-chain antibody (Invention Jian, 08152D) Later, it was stained with streptavidin (Invention, 13025D), and analyzed by flow cytometry (back to Jinson, FACSort). As a control, ICR mice without retaining human chromosomes were also analyzed in the same manner. The results are shown in Figure 22. In the figure, the horizontal axis represents human IgM, and the vertical axis represents CD45R (B220). B cells were labeled with CD45R-positive (FITC) and human IgM-positive (PE) cell groups by 4%, thereby confirming that chimeric mice existed on the cell surface and showed human antibody chains. (Example 46) Human antibody groups were determined from cDNAs derived from spleens of chimeric mice expressing human antibody heavy chains, / C chains, and λ chains. Variation of colonies and base sequences were obtained by Example 29), (Example 23), and (Example 32) Chimeric mice KK15A (from 1-4 strains, shown in Example 10) showing human antibody heavy chain, / C chain, and λ chain, respectively Method production), K2-8 (produced in Example 13), KPG22-2 (produced in Example 31) -91 of the spleen-This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) Bookbinding

1223585 A7 B7 V. Description of the invention (89) RNA, a cDNA synthesized in the same manner as in (Example 5), was subjected to PCR with primers described below to amplify the variable domains of human antibody genes, respectively. As a negative control group, cDNA from the spleen obtained from non-chimeric mouse ICR was used. The undocumented primers in the reference are based on the base sequences obtained from a database such as Genbank. • K15A (heavy chain) for constant field: HIGMEX1-2: CCAAGCTTCAGGAGAAAGT GATGGAGTC ((Sequence 歹 J No. 53) HIGMEX1-1 ···· CCAAGCTTAGGCAGCCAAC GGCCACGCT (Used in the 2nd PCR of VH3BACK) ((Sequence No. 54) OK VH1 / 5BACK (59 ° C, 35 cycles, Marks, etc., Eur · J. Immol ·, 21, 985-, 1991), VH4BACK (59. (:, 35 cycles, Marks, etc., preface, VH3BACK (1st PCR: 59 ° C, 35 cycles, 2nd PCR: 59 ° C, 35 cycles, Morks, etc., preface) • K2-8 (light chain / c) for constant field use ·· KC2H: 5 丨 -CCAAGCTTCAGAGGCAGTTCCAG ATTTC (sequence number 55) Vkl / 4BACK for variable fields (55 ° C, 40 cycles, Marks et al., Eur · J. Immol ·, 21,985-, 1991), Vk2BACK (55 ° C, 40 cycles, Marks et al, preface), Vk3BACK (55t :, 40 cycles, Marks et al, preface) • KPG22-2, (light chain λ) for constant field: C λΜΙχ (the following three primers, to Morrbi, etc.) -92- This paper size applies to China National Standard (CNS) Α4 (210X297 mm)

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Line 1223585 A7 _2! _ 5. Description of the invention (9〇)

IGLl-CR: 5, -GGGAATTCGGGTAGAAGTCACTGATCAG (Serial Number 56)

IGL2-CR: 5 丨 -GGGAATTCGGGTAGAAGTCACTTATGAG (serial number 57)

IGL7-CR ·· 5, -GGGAATTCGGGTAGAAGTCACTTACGAG (sequence number 58) For variable fields: VA1LEA1 (55 ° C, 40 cycles, Williams et al., Eur. J. Immunol., 23, 1456-, 1993), V λ2ΜIX ( 55 ° C, 40 cycles, the VA2LEA1, V λ JLEAD in the previous report of Williams, etc. are mixed, etc., V λ3ΜΙχ (55t, 40 cycles, the V in the previous report of Williams, etc. ALEAl, V A3JLEAD, V λ 3BACK4 and other Morrbi mixers) For constant field use X variable field (3 heavy chain, 3 / c chain, 3 λ chain) combination 94 ° C for 15 seconds, Conditions for recooling temperature shown in the variable field primers for 15 seconds, 72 ° C for 20 seconds, and the number of cycles shown in the variable field primers respectively (using Bajin Weima Co., Shama Circulator-9600) Next. In the second PCR of VH3BACK, the combination of the primers of (H1GMEX-1-1 X VI ^ BACK) was used to expand the amplified product of the first PCR. All amplification products. After 1.5% agarose electrophoresis, it was detected by ethidium bromide staining. As a result, in all combinations, amplification products of expected lengths (heavy chain: about 47 ° 0, light chain / €: about 400 °, and light chain: about 5104 °) were detected. . On the one hand, in the negative control group, no specific amplification products were detected at the same location. These amplified products were extracted from the agarose gel by prep. A. gene (Bayor), and then treated with restriction enzymes (heavy-93- This paper is in accordance with China National Standard (CNS) A4 specifications ( 210 X 297 mm) 1223585 A7 _B7 V. Description of the invention (91) Chain: Hindlll-PstI, light chain / c: Hindlll-PvuII, light chain; I: Hindlll-EcoRI), selected from pUC119 (Treasure wine manufacturing) carrier Hindlll, PstI site (heavy chain), Hindlll, HincII site (/ c chain), Hindlll, EcoRI site (λ chain). Insert into the plastid of the amplification product. For the following (asexual reproduction as shown on the right) ’, determine the base sequence of the amplification product using an automated light scanner (Applied Bio System). • HIGMEX1-2 X VH1 / 5BACK: 10 clones • HIGMEX1-2X VH4BACK: 8 clones • HIGMEXl-2 (2nd PCR, HIGMEX1-1) XVH3BACK: 5 clones • KC2H XV / c 1 / 4BACK: 6 clonal breeding lines • KC2HX V / C2BACK: 7 clonal breeding lines • KC2HX V / C3BACK: 4 clonal breeding lines • C λΜΙχ V A1LEA1: 5 clonal breeding lines • C λΜΙχχ V λ2ΜΙχ: 6 asexual breeding lines • CAMIXXV; 13MIX: base sequences obtained from 5 asexual breeding lines, as analyzed by DNASIS (Hitachi Software Engneering), all sequences are from humans, 23 species in heavy chain There are 21 kinds of functional sequences from the start codon to the constant domain without the start codon. When the determined sequences are identical to each other, the variable domain sequences of 17 heavy chains, 11 / c chains, and 12 Λ chains can be identified. (Example 47) Analysis of human antibody gene variable domain bases from cDNA from spleens of chimeric mice expressing human antibody heavy chain, / c chain, and chain separately -94- This paper applies to China National Standards (CNS) ) A4 size (210X297 mm) 1223585 A7 B7 V. Description of the invention (92) The sequence is based on the base sequence determined in (Example 46) (17 asexual lines of heavy chain, 11 asexual lines of / C chain) Lines, 12 asexual breeding lines of the λ chain), and analyze the following points. 1. Specific known reproductive series V gene fragments to be used in each variable domain sequence 2. Specific known reproductive series J gene fragments to be used in each variable domain sequence 3. To be used in heavy chain variable domain sequences Known reproductive series D fragment specific 4. Additional identification in the N domain of the heavy chain variable domain based on the results of 1, 2, 3 5. Determination of the amino acid sequence derived from the base sequence of the variable domain Shown at (Table 19). For 1,2, the equality check with the germline series V and J fragments registered in Genbank was performed by DNASIS and specified. Notation of VH fragment (Cook et al., Nature gentics, 7, 162-, 1994), V / c fragment (Klein et al., Eur. J. Immunol. 23, 3248-, 1993), and νλ fragment (Williams et al., Preface) The family names of each V segment are recorded in the table. For 3, the equality search with DNASIS for the reproductive series D fragments recorded in the report (Ichihara et al., The EMBOJ ·, 7, 13, 4141-, 1988) is determined based on the consistency of more than 8bp as the index. In the table. For DN *, consider the new DNA family fragments reported by (Green et al., Nature Genetics, 7, 13-, 1994). For 4, based on the results of 1 (V), 2 (J), 3 (D), the base sequence not found in any reproductive series is regarded as N-neck-95- Chinese paper standard (CNS) applies to this paper standard A4 size (210 X 297 mm)

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Line 1223585 A7 B7 V. Description of Invention (93) domain. As a result, 11 of the specific 13 types in the D field were observed in the N field, and the average length was 8.7 bp. For 5, DNASIS was used to convert each base sequence into a single amino acid sequence. Only the CDR3 domain is shown in the table. On the right side of the table are the names of the primers used for breeding in various variable fields and the names of the asexual breeding lines.

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Line -96- This paper size applies to China National Standard (CNS) A4 (210X 297 mm) 1223585 A7

V. Description of the invention (94) Table 19 Group V V segment CDR3 i (D) V primer K15A VH1 VH1-S VRSSSWTEYYYYGMDV J6 (DN1) VH43ACK Η4-10 VK1-1S GG rTMVRGLIITD WYFDL J2 (DXP.l) VH1 / 5BACK Η1 · 7 VH1-24 APYSGRFDY J4 (DK1) VH1 / 5BACK Hi-6 VHM6 ERYYGSGSYQDYYYYYGMDV J6 (DXP.1) VHI / 5BACK Hi-2 VHMo GGYSGYEDYYYYGMDV J6 (DK1) VH1 / 5BACK Η1-10 VH2 VH2-5 SYFDW (DXP1) VH4BACK VH3 VH3-21 EGCSGGSCLPGYYYYGMDV J6 (DLR2) VH1 / 5BACK ΗΜ VH3-23 AHGDPYFDY J4 VH1 / 5BACK Η1.3 VH3-23 DADAFDI SGWDY 1 ': J3 VH1 / 5BACK H1-S VH3-23 J4 (DN1 · ) VH3BACK Η3-3 VH3-23 TGFDL J2 VH4BACK Η4-4 VH3-33 EGGYGSVGDYYYYGMDV J6 (DXP.l) VH1 / 5BACK Η1 · 9 VPD-33 GGYSYGYDYYYYGMDV J6 (DXF1) VH3BACK V3-5 VH3-33 GYSSDNY *) VH4BACK Η4 · 9 VH4 VH4-34 RYSSGWYYFDY J4 (DN1 ·) VH4BACK Η4-15 VH4-59 GRIAVASFDY J4 _1 ·) VH4BACK Η4-2 VH4-59 GSGSYFriFDY J4 VH4BACK Η4 ^ 5 K2-8 V / c 1 018- 3 QQHDNLPFT J3 V / c 1BACK Κ1 · 1 018-8 QQYDNXPIT J5 Va: 13ACK Κ1 · 3 013-3 QQHDi ^ TLPFA J3 Va: 2BACK Κ2 · 2 LI QQYNSYPL T J4 VxlBACK Κ1-6 V &lt; 2 Λ17 MQGTOLLT J4 Vvc2BACK κ2-1 A17 MQGTWI D J5 V &lt; 23ACK κ2-5 · V &lt; 3 A27 QQYGSSPTWT Ji V &lt; 3BACK κ3-1 A27 QQYGSSPFT J3 V &lt; 3BACK κ3-4 All QQYGSSPLWT Jl V &lt; 3BACK Κ3-5 All QQYGSSPPWT Jl V &lt; 33ACK κ3 · 6 V &lt; 6 A26-10 HQSSSLPQT Jl V &lt; 2BACK κ2-4 KPG22-2 V λ 1 DPL3 AAWDDSLDVV JC3 VA 1LEA1 HO DPL5 LWOCA1 GTWOS1 4 DPL5 .GTWDSSLSAGW-JC3 VA 1LEA1 L1-6 DPL5 GTWDSSLSAVV JC2 VA 1LEA1 L1-9 DPLS QSYDSSLSGVV _ · JC3 Va 1LEA1 LU3 VA 2 DPL10 CSYAGSSTLV JC2 V λ 2MIX L24 DPLI1 SSYTSSV2 J2 λ 2MIX L20 DPLil CSYTSSST7V JC2 V λ 2Ν · ΠΧ L2-7 DPL12 SSYAGSNNLV JC3 V λ 2ΜΙχ L2-5 DPL12 SSYAGSNNFVV JC3 V λ 2ΜΙχ L2-6 VA3 DPL16 NSRDSSGNTV JC2 V λ 3cVn China-applicable National Standard (CNS) A4 specification (210X 297 mm) 1223585 A7 B7 V. Description of the invention (95) (Example 48) TT2 (or TT2F) antibody gene of ES cells (heavy chain, light chain / 0 target Carry Preparation of the mouse antibody gene (heavy chain, light chain / C) The human chromosome 14 fragment (Example 9) labeled with the G4 18 resistance gene and the human chromosome 2 labeled with the ploromycin resistance gene (Example 18) or chromosome 22 (Example 35) is introduced into the damaged TT2 (or TT2F) cells. Using such a mouse antibody gene (heavy chain, light chain / c) gene introduced into human 14 + 2 or human 14 + 22 chromosome to destroy TT2 (or TT2F) ES cells, (heavy chain + / C chain: Example 19, heavy chain + λ chain: chimeric mouse prepared by the method of Example 36), mainly, heavy chain and light chain can be expected to produce human-derived antibodies. The abbreviations and the like of the restriction enzymes shown in Figures 23 to 27 are shown below, as follows. Limit S: Kp: Kpnl, B: BamHI, RI: EcoRI, RV: EcoRV, N: Notl, SII: Scall, Sea: Seal, Sfi: Sfil, Sm: Smal, X: Xhol, SI: Sail, dKp: The deletion of Kpnl, (X) ···; Xhol cut-off site of the carrier.

Dotted line part: pBluescriptSKII (+) plastid DNA: LoxP sequence 歹 J 1. Production of LoxP-pstNE0 plastid with LoxP sequence inserted on both sides of the G418 resistance gene. After the antibody gene of TT2 (or TT2F) cells has been knocked down, To remove the G418 resistance gene, two ends of the G41 8 resistance gene (Example 1) can be inserted in the same direction as recombinases (Sauer et al., Proc. Natl. Acad. Sci. USA, 85, 5166-, 1988). Necessary to identify the LoxP sequence (Sauer et al., Preface). The pstNEO group-98 was cut out by pSTneoB plastid DNA (Example 1) with restriction enzyme Xhol-This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1223585 A7 _____ B7 V. Description of the invention (96 ) After purifying the DNA fragment by agarose gel electrophoresis, both ends were smoothed by T4DNA polymerase (pure end kit made by Takara). The plastid DNA pBS246 (plasmid PBS246, a 1 × χ2 cassette vector, U.S. Patent No. 4995317) with LoxP sequence was purchased from GIBCOBRL. The Xhol linker DNA was inserted into the EcoRI and Spel cut sites of this plastid, and changed to the Xhol recognition sequence. The patNEO DNA fragment described above was inserted into this EcoRV cut site of pBS246 to obtain the plastid LoxP-pstNEO (Figure 23). 2. Isolation of TT2 (or butyltin 2F) from a clone of genomic DNA containing C57BL / 6 antibody heavy chain Cy (IgM constant domain) and light chain J / cC / c (Ig / c linkage domain and constant domain) ) Cells were derived from C57BL / 6 mice and CBA mice from F1 mice, and genomic DNA clones derived from C57BL / 6 mice were used to prepare vectors for antibody gene blasting. The genomic DNA library uses the lambda DNA gene library from Clontech's mature C57BL / 6N male liver. As a probe for screening, the following synthetic DNA sequence (60-mer) was used. Heavy chain C # probe: SLACC TTC ATC GTC CTC TTC CTC CTG AGC CTC TTC TAC AGC ACC ACC GTC ACC CTG TTC AAG-3 '(SEQ ID NO: 59) Light chain / c probe: 5, -TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATC CAG TGA GCA GTT AAC ATC TGG AGG-31 (sequence number 60) Analyze the solitary λ clones and include the heavy chain C // or the light chain J / c -C / c The DNA fragment was sub-selected in pBluescript SKII (+) plastid (Stratagene -99- This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 1223585 A7 B7 V. Description of the invention (97) company) (Heavy Chain C: Figure 24; light chain J / cC / c · Figure 25). Using these DNA fragments, a target vector for mouse antibody gene destruction in TT2 (or TT2F) ES cells was prepared as follows. 3. Preparation of mouse antibody heavy chain gene carrier plastids In the field encoding C / ζ containing genomic DNA in the constant region of the mouse antibody heavy chain prepared in 2, will contain the second to fourth expression sequences The DNA fragment (BamHI ~ Xhol) was replaced with LoxP-pstNEO gene (Figure 26) made in 1. The transcription direction of pstNEO is in the reverse direction to the transcription direction of the antibody gene. This plastid DNA was amplified by E. coli TM109 and purified by cesium chloride equilibrium centrifugation (Introduction to Cellular Engineering Experiment Operations, 1992 Kodansha). The purified plastid DNA was cut in one place by the restriction enzyme SacII and used for transfection of TT2 (or TT2F) ES cells. The morphogenetic genomic DNA for the asexual breeding system in which the antibody heavy chain part recombined with the target vector was detected in ES cells from the morphogenic transformant TT2 (or TT2F) ES cells was used as a probe for the Southern Aspiration A DNA fragment (approximately 500 base pairs) that exists in the switch domain upstream of C / ζ. This DNA fragment was obtained by PCR-amplifying 129 mouse genomic DNA under the following conditions. Induction primer: 5, CTG GGG TGA GCC GGA TGT TTT G-3, (sequence number 61) Anti-induction primer: 5, CCA ACC CAG CTC AGC CCA GTT 03, (sequence number 62) Template DNA: EcoRI digestion 129 small Mouse genomic DNA 1 μg reaction buffer, deoxyribonucleic acid mixture, TaqDNA polymerase

Produced by Takara. -100- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) binding

Line 1223585 A7 ___ B7 V. Description of the invention (98) Reaction conditions: 94 degrees C, 3 minutes, 1 time-&gt; 94 degrees C, 1 minute; 55 degrees C, 2 minutes; 72 degrees C, 2 minutes; 3 times -&gt; 94 degrees C, 45 seconds; 55 degrees C, 1 minute, 72 degrees C, 1 minute; 36 times of amplified DNA such as Genbank's database, confirm to limit the enzyme Hindlll, cut one place, subselect EcoRV cut sites in pBluescript plastids. This plastid DNA (S8) was cut with the restriction enzymes BamHI and Xhol, and a PCR fragment (about 550 base pairs) was purified by agarose gel electrophoresis as a probe. The genomic DNA of the TT2 (or TT2F) ES cells transformed with the target vector was digested with the restriction enzymes EcoRI and Xhol, separated by agarose gel electrophoresis, and then subjected to the southern aspiration method, which was detected by the probe described above. 4. Preparation of vector for mouse antibody gene light chain k destruction The DNA fragment (EcoRI ~ SacII) of the J domain (J1 ~ J5) containing genomic DNA in the mouse antibody light chain / c J domain and constant domain prepared in 2 ) And LoxP-pstNEO gene replacement made in 1 (Figure 27). The transcription direction of pstNEO is the same as that of the antibody gene. This plastid DNA was amplified using coliform JM109 and purified by cesium chloride equilibrium centrifugation. The purified plastid DNA was cut in one place by the restriction enzyme κρηΙ and used for transfection of TT2 (or TT2F) ES cells. The morphogenetic genomic DNA for the asexual breeding system in which the antibody heavy chain part recombined with the target vector was detected in ES cells from the morphogenic transformant TT2 (or TT2F) ES cells was used as a probe for the southern aspiration method. Light chain J / cC / C genomic DNA (refer to Figure 25) 3 'end DNA fragment (Xhol ~ EcoRI; about 1.4k base pairs). The genomic DNA of the TT2 (or TT2F) ES cells transformed with the target vector was restricted by digestion of S.EcoRI and Notl. After separation by agarose gel electrophoresis, Southern blotting method was detected, and the detection was -101-This paper is applicable to this paper China National Standard (CNS) A4 specification (210 X 297 mm) 1223585 A7 B7 V. Description of invention (99) The probe described above. (Example 49) Acquisition of mouse ES cell antibody heavy chain gene disruption strain To obtain a similar recombinant of the antibody heavy chain gene, a vector line of the antibody heavy chain labelled with (Example 48) -3 was produced using the restriction enzyme SacII (made by Takara Shuzo). The morphology was introduced into mouse ES cells TT2F according to the method of (Sangshin Shinichi, Biological Manipulation Series 8, Gene Target, Yangtusha, 1995). TT2F cells were treated with trypsin, suspended in HBS to make 2.5 x 107 cells / ml, 5 micrograms of DNA was added, and a gene pulser (Biaure, no-junction resistor unit) was used for electroporation. A 4 mm long cell was used to impinge a capacity of 960 and a voltage of 250 V at room temperature. The cells treated by electroporation were suspended in 20 ml of ES culture medium, and the added cells were seeded in two 100-mm plastic culture dishes (colin) for tissue culture. Similarly, experiments using 10 and 15 micrograms of DNA were performed. After 1 day, it was replaced with G418 (GENETICIN, Xi Goma) containing 300 µg / ml. Seven to nine days later, 176 colonies were picked up and grown on a 12-well plate to a confluent culture, and 4/5 of them were suspended in 0.2 ml of a preservation medium (ES medium + 10% DMSO &lt; Xi Goma>), stored frozen at -80 ° C. The remaining 1/5 was seeded on a 12-well gel-coated plate, cultured for 2 days, and genomic DNA was obtained by the method shown in (Example 2). The genomic DNA of these G418-resistant TT2F cells was digested with the restriction enzymes EcoRI and Xhol (made by Takara Shuzo), separated by agarose gel electrophoresis, and then subjected to the southern aspiration method, which was detected by the probe shown in (Example 48) -3. The same recombinant. As a result, 3 of 176 strains were the same recombinant. The analysis results of the southern aspiration method of wild-type TT2F cells and the same recombinants # 131 and # 141 are shown in Fig. 28 on the left side. In wild-type TT2F cells, digested with EcoRI and Xhol, detected -102- This paper size is applicable to China National Standard (CNS) A4 (210X297 mm) binding

Line 1223585

2 endless belts (a, b). In the same recombinant, any of these orbital bands disappeared, and it is predicted that the orbital band (c) will reappear in the lower part. In the picture # 131, # 14l, the circle of a disappears, and the circle of c reappears. The left side of the figure indicates the size of the DNA. That is, these asexual breeding lines are disrupted by similar recombination of alleles in a single aspect of the antibody heavy chain gene. (Example 50) Antibody heavy chain similar recombinant £ 8 cells were obtained as chimeric mice from frozen stock (Example 49). The antibody heavy chain similar recombinant TT2F cell line # 131 was obtained by ICR or MCH. (ICR) (Keliya Company, Japan) 10 to 12 embryos are injected into 8-cell embryos obtained from male and female mating. The cells were cultured overnight in ES cell culture medium (Example 9), and after placental cells occurred, uteri of parental ICR mice (Benkeria) were temporarily borrowed 2.5 days after pseudopregnancy treatment. About 10 embryos were injected. As a result of transplantation of 94 embryos, 22 daughter mice were born. Chimeric individuals are judged by whether the coat color can be identified as a wild color (strong tea) from TT2F cells in white from the host embryo (ICR). Of the 22 born animals, the wild-colored part showed obvious wild-colored parts, that is to say, there were 8 individuals with Es cell contribution. Among them, more than 80% of the hair color of the 16 individuals were wild-colored female chimeras from es cells. From this result, it was confirmed that the recombinant ES cell line # 3 of the same antibody heavy chain retained the chimerism forming ability. Many of the obtained chimeric mice are female mice exhibiting a very high contribution rate, so there is a high possibility that ES cells will differentiate into functional germ cells (eggs). Among the chimeric mice, the mating results of the female chimeric 2 individuals showing a 100% contribution rate with MCH (ICR) male mice, and the child mice born all showed wild colors. These daughter mice are from # 丨 3 i (refer to Example 42), and it can be considered that the damage is conveyed in a ratio of 1 out of 2 103- This paper size applies the Chinese National Standard (CNS) A4 specification (210 X (297 directors)

Antibody heavy chain alleles. (Example 51) A double-destructed strain obtained from an antibody-like heavy-chain-like recombinant was reported at the unilateral allele by the insertion of the 4184181 resistance gene. Screening of high-concentration G418-resistant strains obtained from G41 8-concentration culture may yield strains with alleles disrupted on both sides (Sangshen Shen- [Biomanipulation Series 8, Gene Target, Yangtusha, 1995). Based on this, we performed the following experiments in order to obtain disrupted strains of alleles on both sides of the same recombinants # 131, # 141 of the heavy chain of the TT2ρ antibody. First, for the two strains # 丨 3 丨 and # 141, in order to determine the lethal concentration of 418, each 1-35 ml culture dish (in this example, the vegetative cells were treated with maidomycin-free primary G418 resistant primary culture cells, refer to Example 9), about 100 cells were seeded every i, containing G418 (GENETICIN, Xi Goma) of 0, 0.5, 1, 2, 3, 5, 8, 10, 15, 20 mg / ml, respectively. The medium was cultured for 10 days. As a result, a clear colony was confirmed at a concentration of 3 g / l, but no colony was confirmed at 5 mg / ml. Based on this result, the minimum lethal concentration was determined to be 5 mg / ml, and 18-tolerant strains were selected at each concentration of 4, 5, 6, 7, 8 mg / ¾ liter. For each of # 131, # 141, in a total of 10 100-mm culture orphans, each of them was seeded with about 106 cells, and the medium containing 0418 of each concentration was used (5 stages of each concentration of 2 in each culture dish). A) training. Twelve days after the start of the culture, pick up the obvious colonies (# 131 ·· 12 strains, # 141 · · 10 strains) in 8 mg / ml petri dishes' as in (Example 49) and cold-bundle for DNA acquisition. . The genomic DNA of these high-concentration G4 18 resistant strains were digested with the restriction enzymes EcoRI and Xhol (made by Takara Shuzo), separated by agarose gel electrophoresis, and then subjected to Nanfang-104. 210 X 297 mm) 1223585 A7 B7

Aspiration method, strains with alleles disrupted on both sides were detected using the probe shown in (Example 48) -3. As a result, it was obtained that the # 131 strain (# 131 · 3) was a strain with both alleles disrupted. The analysis results of the southern sucking method from 6 strains of # 131 are shown (Fig. 28). In wild-type TT2F cells, two wild-type loops (a, b) were detected by Ecori and Cano1 digestion. The same recombinant (# 131 '# 141)' on the one-sided allele disappeared and the c-band a reappeared (Example 49). In addition, by destroying the alleles on both sides, it can be predicted that the wild-type loop b on the other side will disappear, and only the disruptive loop (b) will be lost. In the asexual breeding line 3 (# ΐ3ι · 3) in the figure, observe this loop pattern. That is, this asexual breeding line is the allele disruptor of both sides of the antibody heavy bond gene. (Example 52) The G4 18 resistance marker gene was removed from the antibody heavy chain deletion homozygote TT2F cell line from the antibody heavy chain allele disrupting strain (high concentration G418 resistant strain # 13) obtained in (Example 5 1). The G41 8 resistance marker genes of 1-3) were removed by the following procedure. The expression vector pBS185 (BRL), a Cre recombinase gene containing site-specific recombination between the ιχχρ sequences (Example 48-1) inserted on both sides of the G418 tolerance marker gene, was used in conjunction with caution, biological manipulation Series 8, Gene Target, Yangtusha, 1995, and Takajin Seiki, etc., separate volume of experimental medicine, basic technology of immunology research, P255-, 1995, Yangtusha), introduced # 13 1-3 strains. # 13 1-3 cells were treated with trypsin, suspended in HBS to make 2.5 × 107 cells / ml, 30 mg of pBsl85DNA was added, and a gene pulser (Biaure, without a resistor unit) was used for electricity Kongfa. A voltage of 96V with a capacity of 210V was applied to a 4 mm long electroporation chamber (Example 丨). The cells treated by electroporation were suspended in 5 ml of ES medium, and then feeder cells were used. -105- This paper size is in accordance with China National Standard (CNS) A4 (210X 297 mm).

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Line 1223585 A7 B7 V. Description of the invention (103) Seeded on a 60 mm plastic culture dish (colin) for tissue culture. After 2 days, the cells were treated with trypsin, and the feeder cells were reseeded in 3 100 mm culture m. 100, 200, and 300 cells were used for each culture. The same is done under the condition that only the setting of the gene pulser is changed (connecting the resistor element, the resistance value is infinite). After 7 days, 96 colonies were picked up and divided into two after trypsin treatment. The feeder cells were seeded in two 48-well plates and 48-well plates that were only treated with gelatin. The latter was cultured in a medium containing 300 micrograms / ml of G418 (GENETICIN, Xi Goma) for 3 days, and its survival rate was judged as G418 tolerance. As a result, six asexual clones died in the presence of G418. These G418 susceptible strains were propagated to a confluent culture in a 35 mm culture m, 4/5 of which were suspended in 0.5 ml of storage medium (ES medium + 10% DMSO &lt; Xi Goma &gt;), frozen at -80 ° C save. The remaining 1/5 was seeded on a 12-well gelatin control plate, cultured for 2 days, and genomic DNA was obtained by the method shown in (Example 2). The DHA of these G418 susceptible TT2F cell lines was digested with the restriction enzyme EcoRI (made by Takara Shuzo), separated by agarose gel electrophoresis, and then subjected to the Southern blot method. Needle A) G418 tolerance gene was removed. As a result, the probe A and the hybridized ring band observed in # 131-3 were not detected in the susceptible strain at all. From these results, it was confirmed that the G4 18 tolerance marker gene was removed from the obtained G4 18 susceptible strain. In addition, in the same way as above, the probe B of pBS185DNA was digested with EcoRI and analyzed by the southern suction method. Since no specific loops of probe B and hybridization were detected in these G4 1 8 receptors, Therefore, it is considered that pBS185 containing Cre recombinase is not inserted into the chromosome of the susceptible strain. That is, these susceptible strains can be bound by Example -106- This paper size applies the Chinese National Standard (CNS) A4 specification (210X 297 mm)

Line 1223585 A7 B7 V. The antibody light chain shown in (104) 48-4 of the invention description (loxP sequence exists on both sides of the G4 18 resistance gene) was transformed. (Example 53) Introduction of human chromosome 14 (antibody heavy chain) into an antibody heavy chain-deficient ES cell line A mouse ES cell line (derived from TT2F, G41) lacking the intrinsic antibody heavy chain obtained in (Example 52) 8 susceptibility), as shown in (Example 9), the human chromosome 14 (containing antibody heavy chain gene) labeled with the G41 8 resistance gene was introduced by the microcell method. The obtained G418 resistant strain was analyzed by PCR and the like (Example 9) to confirm that the human chromosome 14 (fragment) containing the human antibody heavy chain gene was retained. (Example 54) The human heavy chromosome 14 fragment-deficient antibody heavy chain ES cell line was introduced into the human second chromosome fragment or the 22nd chromosome from the human heavy chromosome 14 antibody fragment retaining chain obtained in (Example 53) In a mouse ES cell line lacking resistance (G41 8 resistance), a human chromosome 2 fragment (containing an antibody light chain / c) labeled with a ploromycin resistance gene as shown in (Examples 18 and 35) was prepared by the microcell method. Gene) or human chromosome 22 (containing the antibody light chain lambda gene). From the obtained proxomycin and 18 dual-drug-resistant strains' by PCR analysis and the like (Examples 18 and 35), it was confirmed that the human 14th chromosome (fragment) and the 2nd chromosome fragment or the 22nd chromosome (fragment) were retained. (Example 55) A human antibody heavy chain-containing chromosome 14 (fragment) containing an intrinsic antibody heavy chain deletion mouse ES cell was created and a mouse obtained from (Example 53) retained a human antibody-containing heavy chain. The 14th chromosome of the chain gene (body) has intrinsic antibody heavy chain gene-deficient mouse ES cell lines. Chimeric mice were prepared by the method shown in (Example 10). Chimera obtained here

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Line -107-

1223585 A7 B7 V. Description of the invention (105) The human antibody heavy chain produced by B cells from the ES cell line was detected by the method shown in (Example 14). In addition, since the antibody heavy chain gene derived from the B cell of the ES cell is only a human-derived substance introduced into the chromosome, many B cells from the ES cell line produce human anti-weight chains. (Example 56) Intrinsic antibody heavy chain deletion mouse ES cells from retained human chromosomes 14 + 2, 14 + 22 (fragment) were made into chimeric mice. Retained human cells obtained from (Example 54) 14 + 2nd, 14 + 22nd chromosome (fragment) intrinsic antibody heavy chain gene deletion mouse ES cell strains were made into chimeric mice according to the methods shown in (Examples 19, 36) and the like. The chimeric mice obtained here were human antibody heavy chains and light chains / C or light chains derived from B cells of the ES cell line; I, were detected according to the methods shown in (Examples 14, 23, 32). Also, as in (Example 55), the B cell derived from the ES cell, the functional antibody heavy chain gene, is only a human-derived substance introduced into the chromosome, so many B-cells derived from the ES cell produce human Antibody heavy chain. In addition, as shown in (Examples 37 and 38), both the heavy chain and the light chain detected complete human antibody molecules derived from humans. (Example 57) A human antibody-producing hybridoma was obtained from a chimeric mouse derived from a human ES cell that retains the intrinsic antibody heavy chain deletion mouse 14 + 2, 14 + 22 chromosome (fragment) (Examples 15, 25, and 34) Similarly, the chimeric mice prepared in (Example 56) were immunized with the target antigen, the spleen was removed, and the cells were fused with myeloma tumor cells to form hybrid tumors. After 1 to 3 weeks of culture, the culture supernatant was analyzed by ELISA. ELISA method (Examples 14, 15, 21, 24, 25, -108- this paper size is applicable to @ B 家 标准 (CNS) M specification (2iG χ 297 directors) binding)

Lines 1223585 A7, B7 V. Description of the invention (106) 33, 34, 37, 38) were carried out to obtain human antibody-positive and human antibody-positive and immunized antigen-specific clones. (Example 58) Obtaining an antibody light chain gene-disrupting strain from an antibody heavy chain deletion homozygote mouse ES cell The antibody heavy chain deletion homozygote TT2F cell strain (G4 18 sensitivity) obtained in (Example 52) The same recombinants that disrupted the antibody light chain gene were obtained in the following order. The antibody light chain tag vector prepared in (Example 48-4) was used to introduce the above-mentioned TT2F in accordance with the method of (Shoichi Shinichi, Biological Manipulation Series 8, Gene Target, Yangtusha, 1995) at a linear ratio of the restriction enzyme KρηΙ (made by Takara Shuzo) Cell line (G418 sensitivity). After 7 to 9 days, the born colonies were picked up and frozen in accordance with the method shown in (Example 49) to obtain genomic DNA. The genomic DNA of the G418 resistant strain was digested with the restriction enzymes EcoRI and Notl (made by Takara Shuzo), separated by agarose gel electrophoresis, and analyzed by the Southern blot method. The same recombinant was detected with the probe shown in (Example 48-4). . (Example 59) Obtaining a double-disrupted strain from an antibody light chain similar recombinant. For the TT2F antibody light chain similar recombinant obtained in (Example 58) (and the antibody heavy chain lacks a homologous conjugation), light was obtained in the following order. Destroyed strains of alleles on both sides of the strand. A high-concentration G418-resistant strain was obtained in the same manner as in (Example 5 1), and frozen and stored to obtain DNA. The genomic DNA of a high-concentration G418-resistant strain was digested with the restriction enzymes EcoRI and Notl (made by Takara Shuzo), separated by agarose gel electrophoresis, and then subjected to the southern aspiration method. Two samples were detected by the ink dot method shown in Example 48-4 Lateral allele disrupted strains. (Example 60) A homologous conjugation of an antibody light chain deletion (and an antibody heavy chain deletion are the same as -109-) This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (107) source zygote) TT2F cell line removal of the G4 18 resistance marker gene (Example 59) The allele-deficient strain (high concentration G418-resistant strain) on both sides of the antibody light chain obtained (Example 59) G418 tolerance marker genes were removed in the order shown in (Example 52). The expression vector pBS185 (BRL) containing a Cre recombinase gene that had site-specific recombination inserted between the IοχP sequences (Example 48-1) flanking the G4 18 resistance marker gene was performed according to the method of (Example 52). , Introduced in the strain described above. In the same manner as in Example 52, the obtained G418 susceptible strain was propagated to a confluent culture in a 35 mm culture m, and 4/5 of the suspension was suspended in 0.5 ml of a storage medium (ES medium + 10% DMSO &lt; Xige Horses>), stored frozen at -80 ° C. The remaining 1/5 was seeded on a 12-well gelatin-coated plate, cultured for 2 days, and genomic DNA was obtained by the method shown in (Example 2). The genomic DNA of these G4 1 8 susceptible TT2F cells was digested with the restriction enzyme EcoRI (made by Takara Shuzo), separated by agarose gel electrophoresis, and then subjected to the southern aspiration method. The 3.2kbXhoI fragment from pSTneoB containing the G418 resistance gene was used as a probe. To confirm removal of the G418 tolerance gene. (Example 61) Introduction of human chromosome 14 (antibody heavy chain) into an antibody heavy and light chain-deficient ES cell line Mouse obtained from (Example 60) mice lacking both intrinsic antibody heavy and light chains In the ES cell line (derived from TT2F, G418 sensitivity), the human chromosome 14 (containing the human antibody heavy chain gene) which was labeled with the G41 8 resistance gene as shown in (Example 9) was introduced by the microcell method. The obtained G4 18 resistant strain was analyzed by PCR and the like (Example 9) to confirm that the human chromosome 14 (fragment) containing the human antibody heavy chain gene was retained. (Example 62) The deletion of the heavy and light chains of the antibody and the retention of human chromosome 14 -110- This paper size applies the Chinese National Standard (CNS) A4 (210X 297 mm) binding

Line A7 B7 V. Introduction to the invention (ι〇8) (antibody heavy chain) ES cell line into human chromosome 2 (light chain ")

In the mouse ES cell strain (derived from TT2F, G418 tolerance) of the intrinsic antibody heavy and light chain deletions obtained in (Example 6 1) and retaining human chromosome 14, as shown in (Example 18), The cell method was used to introduce a partial fragment of the human chromosome 2 (containing the human antibody light chain &amp; gene) of the ploromycin resistance gene. With respect to the obtained dual drug-resistant strains of pramycin and G418, the retention of the human chromosome 14 (fragment) and the second chromosomal fragment was confirmed by pCR analysis and the like (Example 18). (Example 63) Introduction of human chromosome 22 (light chain λ) in (ESC 61) into an ES cell line in which the heavy and light chains of the antibody were deleted and the human 14th staining ㈣ (antibody heavy chain) was retained The human ES cell line (from TT2F, G418 tolerance) in which the heavy and light chains of the antibody were deleted and the human chromosome 14 was retained (as shown in Example 35). Introduction of chromosome 22 (containing human antibody light chain; gene). From the obtained protomycin and G418-tolerant strains by PCR analysis (Example 35), it was confirmed that human chromosome 14 (fragment) and chromosome 22 (fragment) were retained. (Example 64) Human antibody 2 (antibody light chain / c), 14 (antibody heavy chain), 22 (antibody lambda chain) chromosome or partial fragment intrinsic antibody heavy chain, light chain deletion mouse were simultaneously retained Obtaining ES cells To obtain mouse ES cells that simultaneously retain three human chromosomes, human chromosomes 2 or 22 were inserted into embryomycin (7, and only 4 inches of tolerance (Izumi et al. 'Exp · Cell · Res ·, 197, 229, 1991), hygromycin resistance (Wind et al., Cell, 82, 321-, 1995) and other marker genes. This was performed according to the method shown in (Examples 16, 26). Intrinsic -111 obtained in (Example 62)-This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) A7 B7 V. Description of the invention (109 L ^ Several heavy chain and light chain missing phantoms The mouse ^ cell line (derived from TT2F, G418 resistance, Puro, and Su resistance) of the human chromosome 14 (fragment) and the second chromosome fragment was retained in the same manner as shown in (Example 9) Introduced human chromosome 22 (containing human 基因 m chain and gene) labeled with embryomycin resistance or hygromycin resistance For feeder cells for ES cell culture, 5 individual selection standards are used. In the case of a hygromycin resistance marker, the primary cultured fibroblasts obtained from the expressed transfer gene system are used. Johns On et al., Nucleic palpitations 23, No. 7, 1273-, 1995). The obtained G418, puramycin, hygromycin (or embryomycin) double-tolerant strains retain the three human chromosomes described above (Fragment) Confirmed by PCR analysis, etc. (Examples 9, 18, 35). Obtained in (Example 63), the intrinsic antibody heavy chain and light chain are deleted, and human chromosome 14 (fragment) and chromosome 22 are retained Some fragments of mouse Es cell lines (derived from TT2F, G41 8 resistance, and protoxin resistance) were also similarly introduced into human chromosome 2 fragments labeled with a gene of hygromycin or embryomycin resistance. 65) Self-retaining the intrinsic antibody heavy chain and light chain gene-deficient mouse ES cells containing human antibody gene (heavy key + light chain) chromosome (fragment) contains self-retaining chimeric mice (Examples 61, 62, 63, 64) Chromosomes of human antibody genes obtained Fragment) Intrinsic antibody heavy chain and light chain gene-deficient mouse ES cell strains were created as post-combined mice by the methods shown in (Example 10). The chimeric mice obtained here were obtained from the host The mouse antibodies produced by the B cells of the embryo and the human antibodies mainly produced by the B cells from the ES cell line were detected by the method shown in (Examples 14, 23, 32). The paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1223585 A7 B7 V. Description of the invention (110) B-cell functional antibody heavy chain and light chain / c gene are only for introduction from the chromosome Human things. Therefore, many B cells derived from ES cells produce human antibody heavy and light chains / c (Lonberg et al., Nature, 368, 856-, 1994). In addition, by the methods shown in (Examples 37 and 38), both human and light chain-derived human antibody molecules were detected. (Example 66) A human antibody-producing hybrid was obtained from a chimeric mouse derived from an ES antibody-containing mouse heavy chain and light chain gene-deleted mouse chromosome (fragment) containing a human antibody gene (heavy chain + light chain). The tumor was immunized with the target antigen in the chimeric mouse obtained in (Example 65) in the same manner as in Example 25, and the spleen was removed and fused with myeloma tumor cells to form a hybrid tumor. After 1 to 3 weeks of culture, the culture supernatant was analyzed by ELISA. The ELISA method was performed according to the methods shown in (Examples 14, 15, 21, 22, 23, 24, 25, 33, 34, 37, 38), and human antibody-positive and human antibody-positive and immune-specific asexual antibodies were obtained. Breeding line. (Example 67) Chimeric mice made with host embryos with heavy chain gene deletions showed wild colors in the offspring of chimeric mice derived from intrinsic antibody heavy chain flanking allele deletion TT2F cell strains prepared in (Example 49) Alternatively, individuals who retain the missing alleles are selected by analysis of the southern suction method or PCR (Example 49), etc. (the probability of expectation is 1/2). For the mice born from the mating of the male and female individuals with these antibody heavy chain deletion heterozygotes, Southern blot analysis or PCR (Example 49) was performed to select individuals who retained the deleted alleles (expected probability). Is 1/2). For the mice born from the mating of male and female individuals with these antibody-heavy heterozygous junctions, the Southern suction method (see Example 49) was performed to show the μ chain appearance on the surface of lymphocytes (Kitamura et al., -113- This paper size applies to China National Standard (CNS) A4 (210X297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (111)

Nature, 35 0, 423-, 1991), etc., it can be obtained that the two sides of the allele are deleted, and the functional heavy antibody of the antibody itself can hardly produce the heavy chain deletion of the homozygous conjugation (the expected probability is 1/4, in Membrane type #chain deletion mice results: See Kitamura et al., Nature, 350, 423-, 1991). Embryos obtained from the mating of homozygous female and male individuals reared in a clean environment can be used as hosts for chimeric mice. In the chimeric mice in this case, most of the functional B cells came from the injected ES cells. RAG-2 deficient mice (Shinkai et al., Cell, 68, 855-, 1992), etc., and other mouse systems that cannot produce functional B cells themselves can also be used for this purpose. In this system, used in (Examples 62, 63, 64), it can be obtained that the intrinsic antibody heavy chain and light chain are deleted and the human 14 + 2 or 14 + 22 or 14 + 2 + 2 + 22 is retained Chromosome (fragment) mouse ES cell lines were used to prepare chimeric mice by the methods shown in (Example 10) and the like. In the obtained chimeric mice, functionally human antibody heavy chain (on chromosome 14), light chain (on chromosome 2), and light chain λ (on chromosome 22) genes were derived from ES cells in B cells. It mainly produces antibodies made from human heavy chains and human light chains. (Example 68) The retention of human chromosomes in chimeric mouse offspring from ES cells introduced into the human chromosome 14 (fragment) will be retained in the same manner as in (Example 9), and it will be advantageous to use (Example 9 ) The mouse ES cells were replaced with TT2F (39, X0, Example 39). Chimeric mice of human chromosome 14 (fragment) were mixed with wild-type ICR mice (Albino, Japan Klea Corporation). . Regarding the genomic DNA prepared from the tail of the born wild-colored daughter mouse, the retention of the human 14th chromosome fragment was examined by PCR (see Examples 9, 42, 43). Human 14th Dye Reserve -114- This paper size applies to China National Standard (CNS) Α4 size (210 × 297 mm) binding

Line 1223585 A7 B7 V. Description of the invention (112) A mouse ES cell line of a chromosome (fragment), as shown in (Example 42) and (Example 43), differentiates into a functional egg in a chimeric mouse or Sperm, from which descendants can convey human chromosome 14 (fragment). That is, a viable mouse system retaining the chromosome 14 (fragment) containing the human antibody heavy chain gene can be established. In this example and the following examples 69 to 74, the human chromosome 14 (fragment) refers to the gene containing human antibody heavy chain, the human chromosome 2 (fragment) refers to the gene containing human antibody / C chain, and the human chromosome 22 (fragment) ) Refers to genes that contain human antibodies. (Example 69) The retention of human chromosomes in chimeric mouse offspring from ES cells introduced into the human chromosome 22 (fragment) will be retained using the same method as in (Example 30) and using the same method as in (Example 30). Chimeric mice of human chromosome 22 (fragment) obtained by the method of replacing mouse ES cells with TT2F (39, XO) were mated with ICR mice. The genomic DNA prepared from the tails of wild-born mice was examined by PCR for the retention of human chromosome 22 fragments (see Examples 30, 42, 43). The mouse ES cell line retaining the human chromosome 22 or a partial fragment thereof, as shown in (Example 42) and (Example 43), was differentiated into functional eggs or sperm in chimeric mice and derived from its descendants. Can convey human chromosome 22 (fragment). That is, a viable mouse system that retains human antibody-containing light chains; chromosome 22 (fragment) of the I gene can be established. (Example 70) The mating of a human mouse chromosome 2 segment and a 14th chromosome (fragment) at the same time was performed by mating a mouse chromosome segment obtained by retaining (Example 42) or (Example 43) Human chromosome 14 obtained from the mouse system and retention (Example 68) -115- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) binding

Line 1223585 A7 B7 V. Explanation of the invention (113) (fragment) Mouse system mating The genomic DNA prepared from the tail of the child mouse was analyzed by PCR method (Examples 9, 42, 43), and the human 2 Chromosome partial fragments and human chromosome 14 (fragment) individuals. (Example 71) A mouse system that retains human chromosome 22 (fragment) obtained by mating with a mouse individual that simultaneously retains human chromosome 22 (fragment) and chromosome 14 (fragment) (Example 69) and ( Example 68) The genomic DNA prepared from the tails of child mice born from the systemic mating of human chromosome 14 (fragment) -preserving mice was analyzed by PCR method (Examples 30, 42, 43), and human 22 Chromosome (fragment) and human chromosome 14 (fragment). (Example 72) The retained human chromosome 22 (fragment) obtained by mating with a mouse individual that simultaneously retained the human chromosome 2 fragment, the 14th chromosome (fragment) and the 22nd chromosome (fragment) (Example 71) The genomic DNA prepared from the mouse system of the human chromosome 14 (fragment) and the mouse system retaining the human chromosome 2 fragment obtained in (Examples 42 and 43) was genomic DNA prepared by PCR and the like (Examples) (9, 30, 42, 43) analysis, it is possible to keep three human chromosomes of human chromosome 22 (fragment) and human chromosome 14 (fragment) and human chromosome 2 fragment. Or the mouse system for retaining human chromosome 2 (fragment) and human chromosome 14 (fragment) obtained in (Example 70) and the mouse system for retaining human chromosome 22 (fragment) obtained in (Example 69) For mating, it is also necessary to retain individuals with three human chromosomes at the same time. (Example 73) The mating system of a mouse system produced mainly from complete human antibodies was used to produce -116- This paper size applies Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1223585 A7 B7 V. Description of the invention (114) Repeatedly retaining human 2+ chromosome 14th (Example 70), 14+ 22nd (Example 71), 2+ 14th + 22nd (Example 72) mouse system and deletion inherent Sex antibody heavy chain (Example 67; Kitamura et al., Nature, 350, 423-, 1991), light chain / c (Zou et al., EMBO J., 12, 811-, 1993; Chen et al., EMBO J., 12, 821-, 1993) gene mating of mice, selection by PCR analysis, etc. (Examples 9, 30, 42, 43) to select to retain human 2 + 14th, 14th + 22nd, or 2 + 14th + 14th A mouse system with 22 chromosomes was established to produce a mouse system that primarily produces complete human chromosomes (Green et al., Nature Genetics, 7, 13-, 1994, Lonberg et al., Nature, 368, 856-, 1994). (Example 74) Human antibody-producing hybridomas were obtained from a mouse system retaining human chromosomes containing human antibody genes obtained by mating. (Example 25) The mouse chromosome containing the human antibody gene obtained in (Examples 42, 43, 68, 69, 70, 71, 72, 73) was left as the target antigen for immunization, the spleen was removed, and myeloma cell Cells fuse to form a hybridoma. After 1 to 3 weeks of culture, the culture supernatant was analyzed by ELISA. The ELISA method was performed in accordance with the methods shown in (Examples 14, 15, 21, 22, 25, 33, 34, 37, 38), and human antibody-positive and human antibody-positive and immunized antigen-specific clones were obtained.

(Example 75) Detection and quantification of mouse IgM in chimeric stingy sera from allele-damaged TT2F cell lines on both sides of mouse antibody heavy chain Both sides of mouse antibody heavy chain obtained from (Example 51) In the isogenically disrupted TT2F cell line (# 13 1-3), the child mice born by the method shown in (Example 40) were used to bloodize three individuals with chimerism rates of 0%, 50%, and 99%, respectively. -117- This paper size applies to China National Standard (CNS) A4 (210X 297mm) binding

Line 1223585 A7 B7 V. Description of the invention (115) Detection and quantification of mouse IgM in the clear. Blood was collected from chimeric mice at about 2 weeks of age, and the mouse IgM concentration in serum was determined by ELISA (Example 14). An anti-mouse IgM antibody (Kirkegaard &amp; Perry Laboratories, 01-18-03) diluted in PBS was fixed, followed by the addition of 5% FBS in PBS-diluted serum samples. Add peroxidase-labeled anti-mouse IgM antibody (Kirkegaard &amp; Perry Laboratories, 074-1803), use TMBZ as the substrate, measure the absorbance at 450nm, and use purified mouse IgM (invented by 0308ID) as the standard. Characteristically, it was diluted with FBS-added PBS, and the results are shown in Table 20. In chimeric mice derived from mouse TT2F cells with alleles flanked on both sides of the mouse antibody heavy chain, it was confirmed that the chimeric rate was 99%, the IgM concentration was low, and the mouse heavy chain genes of ES cells were almost inorganic. Table 20. Mouse IgM concentration in chimeric mice (ELISA) Chimeric rate% IgM (mg / ml) 0 12 50 11 99 1.5 Industrial applicability According to the present invention, it is provided to retain single or plural foreign chromosomes or fragments thereof 'A chimeric non-human animal expressing the gene on the chromosome or a fragment. By using the chimeric non-human animal of the present invention, a biologically active substance can be produced. According to the present invention, it is provided to retain single or plural foreign chromosomes or their fragments. -118- This paper size applies the Chinese National Standard (CNS) A4 specification (210 × 297 mm) 1223585 A7 B7 V. The invention description paragraph (116) Cells with multipotential potential as expressed by genes on chromosomes or fragments thereof. These cells can be used to treat genetic diseases such as bone marrow transplantation. Sequence table Sequence number: 1 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-strand Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences TGGAAGGTGG ATAACGCCCT 20 Sequence number: 2 Sequence length: 22 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequence TCTTCTCCT CCAACATTAG CA 22 Sequence number: 3 Sequence length: 21 Sequence type: Nucleic acid chain number: Single-strand geometry: Straight-119- This paper standard applies to Chinese National Standard (CNS) A4 Specifications (210X297 mm) 1223585 A7 B7 V. Description of the invention (117) Sequence type: Other nucleic acid synthetic DNA sequences TGTAGGGGAC CTGGAGCCTT G 21 Sequence number · 4 Sequence length: 21 Sequence type: Nucleic acid strand number: Single-strand geometry: Straight Chain

Sequence type: Other nucleic acid synthetic DNA sequences TTGACAACTC ACCTGGACTA G 21 Sequence number: 5 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence CTCTCCTGCA GGGCCAGTCA 20 Sequence number: 6 Sequence length: 22 Sequence type: Nucleic acid chain number: Single-strand geometry: Straight-120- This paper standard applies to China National Standard (CNS) A4 specifications (210 X 297 mm) 1223585 A7 B7 V. Description of the invention (1) Sequence type: Other nucleic acid synthesis DNA sequence TGCTGATGGT GAGAGTGAAC TC 22 Sequence number: 7 Sequence length: 20 Sequence type: Nucleic acid strand number: Single-stranded geometry : Straight chain

Sequence type: Other nucleic acid synthetic DNA sequence AGTCAGGGCA TTAGCAGTGC 20 Sequence number: 8 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence GCTGCTGATG GTGAGAGTGA 20 Sequence number: 9 Sequence length: 20 Sequence type: Nucleic acid chain number: Single-strand geometry: Straight-121-This paper size applies to China National Standard (CNS) A4 specifications (210 X 297 mm)

Order

Line 1223585 A7 B7 V. Description of the invention (119) Sequence type: Other nucleic acid synthetic DNA sequences TGGTGGCTGA AAGCTAAGAA 20 Sequence number: 10 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences CCAGAAGAAT GGTGTCATTA 20 Sequence number: 11 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence TCCAGGTTCT GCAGAGCAAG 20 Sequence number: 12 Sequence length: 20 Sequence type: Nucleic acid chain number: Single-strand geometry: Straight-122- This paper size applies to China National Standard (CNS) A4 specifications (210X297 mm) 1223585 A7 B7 V. Description of the invention (12) Sequence type: Other nucleic acid synthesis DNA sequence TGTAGTTGGA GGCCATGTCC 20 Sequence number: 13 Sequence length: 20 Sequence type: Nucleic acid strand number: Single-strand geometry: Straight Sequence type: Other nucleic acid synthetic DNA sequence CCCCACCCAT GATCCAGTAC 20 Sequence number: 14 Sequence length: 20 Sequence type: Nucleic acid chain number: Single-stranded geometry: Linear sequence type: Other nucleic acid synthetic DNA sequence GCCCTCAGAA GACGAAGCAG 20 Sequence number : 15 Sequence length: 22 Sequence type: Nucleic acid strand number: Single-stranded Geometry: Straight-123- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)

Binding

Line 1223585 A7 B7 V. Description of the invention (121) Sequence type: Other nucleic acid synthetic DNA sequences GAGAGTTGCA GAAGGGGTGA CT 22 Sequence number: 16 Sequence length: 22 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences GGAGACCACC AAACCCTCCA AA 22 Sequence number: 17 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-strand Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences GGCTATGGGG ACCTGGGCTG 20 Sequence number: 18 Sequence length: 22 Sequence type: Nucleic acid Chain number: Single-strand -124- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)

Order

Line 1223585 A7 B7 V. Description of the invention (122) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences CAGAGACACA GGCACGTAGA AG 22 Sequence number: 19 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequence TTAAGGGTCA CCCAGAGACT 20 sequence number: 20 sequence length: 20 sequence type: nucleic acid chain number: single strand geometry: straight chain

Sequence type: other nucleic acid synthetic DNA sequence TGTAGTTGGA GGCCATGTCC 20 sequence number: 21 sequence length: 20 sequence type: nucleic acid strand number: single-strand -125- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) )

Order

Line 1223585 A7 B7 V. Description of the invention (123) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequence CAAAAAGTCC AACCCTATCA 20 Sequence number: 22 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence GCCCTCAGAA GACGAAGCAG 20 sequence number: 23 sequence length: 20 sequence type: nucleic acid chain number: single-strand geometry: linear

Sequence type: Other nucleic acid synthetic DNA sequence TCGTTCCTGT CGAGGATGAA 20 Sequence number: 24 Sequence length: 20 Sequence type: Nucleic acid strand number: Single-chain-126- This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm) 1223585 A7 B7 V. Description of the invention (124) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences TCACTCCGAA GCTGCCTTTC 20 Sequence number: 25 Sequence length: 21 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence ATGTACAGGA TGCAACTCCT G 21 Sequence number: 26 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single strand Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence TCATCTGTAA ATCCAGCAGT 20 Sequence number: 27 Sequence length: 20 | Sequence type: Nucleic acid Chain number: Single-strand -127- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)

Order

Line 1223585 A7 B7 V. Description of the invention (125) Geometry: straight chain

Sequence type: other nucleic acid synthetic DNA sequence GATCCCATCG CAGCTACCGC 20 sequence number: 28 sequence length: 20 sequence type: nucleic acid chain number: single-strand geometry: straight-chain

Sequence type: Other nucleic acid synthetic DNA sequences TTCGCCGAGT AGTCGCACGG 20 Sequence number: 29 Sequence length: 22 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence GATGAACTAG TCCAGGTGAG TT 22 Sequence number: 30 Sequence length: 22 Sequence type: Nucleic acid Chain number: Single-strand -128- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)

Order

Line 1223585 A7 B7 V. Description of the invention (126) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences CCTTTTGGCT TCTACTCCTT CA 22 Sequence number: 3 1 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-strand Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences ATAGAGGGTA CCCACTCTGG 20 Sequence number: 32 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence AACCAGGTAG GTTGATATGG 20 Sequence number: 33 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-strand -129-This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)

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Line 1223585 A7 B7 V. Description of the invention (127) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences AAGTTCCTGT GATGTCAAGC 20 Sequence number: 34 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences TCATGAGCAG ATTAAACCCG 20 Sequence number: 35 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence TGTGAAGGAG GACCAGGTGT 20 sequence number: 36 sequence length: 20 sequence type: nucleic acid strand number: single-strand-130- This paper size applies to China National Standard (CNS) A4 specification (210X 297 mm) ) 1223585 A7 B7 V. Description of the invention (128) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences TGTAGGGGTT GACAGTGACA 20 Sequence number: 37 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences CTGAGAGATG CCTCTGGTGC 20 Sequence number: 3 8 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence GGCGGTTAGT GGGGTCTTCA 20 Sequence number: 39 Sequence length: 20 Sequence type: Nucleic acid strand number: Single-strand -131- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) )

Order

Line 1223585 A.7 B7 V. Description of the invention (129) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences GGTGTCGTGG AACTCAGGCG 20 sequence number · 40 sequence length: 20 sequence type: nucleic acid chain number: single strand geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences CTGGTGCAGG ACGGTGAGGA 20 Sequence number: 41 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequence GCATCCTGAC CGTGTCCGAA 20 Sequence number: 42 Sequence length: 20 Sequence type: Nucleic acid strand number: Single-strand -132- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) ) 1223585 A7 B7 V. Description of the invention (13〇) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences GGGTCAGTAG CAGGTGCCAG 20 Sequence number: 43 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type :. Other nucleic acid synthetic DNA sequences AGTGAGATAA GCAGTGGATG 20 sequence number: 44 sequence length: 20 sequence type: nucleic acid chain number: single-strand geometry: linear

Sequence type: Other nucleic acid synthetic DNA sequence GTTGTGCTAC TCCCATCACT 20 Sequence number: 45 Sequence length: 21 Sequence type: Nucleic acid strand number: Single-stranded-133- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) )

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Line 1223585 A7 B7 V. Description of the invention (131) Geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequences TTGTATTTCC AGGAGAAAGT G 21 Sequence number: 46 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences GGAGACGAGG GGGAAAAGGG 20 Sequence number: 47 Sequence length: 27 Sequence type: Nucleic acid Chain number: Single-stranded Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence ATGGACTGGA CCTGGAGGRT CYTCTKC 27 Sequence number: 48 Sequence length: 27 Sequence type: Nucleic acid strand number: Single-strand -134- This paper size applies Chinese National Standard (CNS) A4 specification (210 X 297 public (Centi) 1223585 A7 B7 V. Description of the invention (132) Geometry: straight chain

Sequence type: other nucleic acid synthetic DNA sequence ATGGAGYTTG GGCTGASCTG GSTTTYT 27 sequence number: 49 sequence length: 27 sequence type: nucleic acid chain number: single-strand geometry: linear

Sequence type: Other nucleic acid synthetic DNA sequences ATGRAMMWAC TKTGKWBCWY SCTYCTG 27 Sequence number: 50 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-strand Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequences CAGAGGCAGT TCCAGATTTC 20 Sequence number: 5 1 Sequence length: 20 Sequence type: Nucleic acid Chain number: Single-strand -135- This paper size applies to China National Standard (CNS) A4 specification (210X297 mm)

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Line 1223585 A7 B7 V. Description of the invention (133) Geometry: straight chain

Sequence type: other nucleic acid synthetic DNA sequence TGGGATAGAA GTTATTCAGC 20 sequence number: 52 sequence length: 20 sequence type: nucleic acid strand number: single strand geometry: straight chain

Sequence type: Other nucleic acid synthetic DNA sequence ATGGACATGR RRDYCCHVGY KCASCTT 27 sequence number: 53 sequence length: 28 sequence type: nucleic acid chain number: single-strand geometry: linear

Sequence type: Other nucleic acid synthetic DNA sequence CCAAGCTTCA GGAGAAAGTG ATGGAGTC Sequence number: 54 Sequence length: 28 Sequence type: Nucleic acid chain number: Single-strand -136- This paper size applies Chinese National Standard (CNS) A4 specification (210X 297 mm )

Order

Line! 223585 A7 B7 V. Description of the invention (彳 34) Geometry: straight chain

Sequence type: other nucleic acid synthetic DNA sequences

CCAAGCTTAG GCAGCCAACG GCCACGCT Sequence number: 55 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single-stranded Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequences

CCAAGCTTCA GAGGCAGTTC CAGATTTC Sequence number: 56 Sequence length: 28 Sequence type: Nucleic acid Chain number: Single strand Geometry: Linear

Sequence type: Other nucleic acid synthetic DNA sequence GGGAATTCGG GTAGAAGTCA CTGATCAG Sequence number: 5 7 Sequence length: 28 Sequence type: Nucleic acid strand number: Single-strand-137- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 public) (Centimeter) 1223585 A7 B7 V. Description of the invention (135) Geometry: straight chain

Sequence type: other nucleic acid synthetic DNA sequences

GGGAATTCGG GTAGAAGTCA CTTATGAG Sequence number: 58 Sequence length: 28 Sequence type: Nucleic acid Chain number: Single strand Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequences

GGGAATTCGG GTAGAAGTCA CTTACGAG Sequence number: 59 Sequence length: 60 Sequence type: Nucleic acid Chain number: Single strand Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequences

CCTGAGCCTC

ACCTTCATCG TCCTCTTCCT

TTCTACAGCA CCACCGTCAC CCTGTTCAAG Sequence number: 60 Sequence length: 60 -138- This paper size applies to Chinese National Standard (CNS) A4 specifications (210X 297 mm) 1223585 A7 B7 V. Description of the invention (136) Sequence type: Number of nucleic acid chains: single Chain geometry: straight chain

Sequence type: other nucleic acid synthetic DNA sequences

TGATGCTGCA CCAACTGTAT CCATCTTCCC ACCATCCA

GT GAGCAGTTAA CATCTGGAGG Sequence number: 61 Sequence length: 22 Sequence type: Nucleic acid Chain number: Single-strand Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequences

CTGGGGTGAG CCGGATGTTT TG Sequence number: 62 Sequence length: 22 Sequence type: Nucleic acid Chain number: Single strand Geometry: Linear

Sequence type: other nucleic acid synthetic DNA sequences

CCAACCCAGC TCAGCCCAGT TC -139- This paper size applies Chinese National Standard (CNS) A4 specification (210X297 mm) Application date i 85. 9. 25 Case No. 089113072 Class Add Μ 7 (12V heart

Replacement of the Chinese manual of the invention (January 1992). Chalk Patent Specification 1223585 Chinese Name of the Animal and its Manufacturing Method. English Name: am ^ ERIC ANIMAL AND METHOD for PRODUCING the SAME. 1. Totsuka Ichika 3. Hanaoka Kazunari 5. Ishida Gongjun Script 2. Yoshida Kyo 4 · Mitsuo Omura Residence and Home Furnishing 1.2.5. Both Mura Linpu ale base plate technology Inside the research institute 3. Saijotsu, Uenohara-cho, Kituryu-gun, Yamanashi Prefecture, Yamanashi, Japan 1469 4. Izumi 70, Yonago, Tottori, Japan, Japan 441 Name (nationality) Nationality Japanese quotient Kirin Barley Co., Ltd. Line Applicants f affairs% Sato Yasuhiro, No. 1Q, Shinkawajichome, Chuo-ku, Tokyo, Japan This paper is sized for China National Standard (CNS) A4 (210 X 297 mm)

Claims (1)

1223585 Patent Application No. 089113072 Chinese Application for Patent Scope Replacement (Jan 1993) VI. Patent Application Scope A8 C8 D8 Bulletin 1. Method for making chimeric mice with foreign chromosomes or fragments thereof The purpose is to produce microcells containing foreign chromosomes or fragments thereof that encode regions of human antibody genes, and by fusion with the microcells, transfer the foreign chromosomes or fragments thereof into cells with multipotential potential. 2. The method according to item 丨 of the scope of patent application, wherein the total base length of the foreign chromosome or a fragment thereof is 1 Mb (million base pairs) or more. 3. The method according to item 丨 of the patent application scope, wherein the microcells containing the foreign chromosome or a fragment thereof are derived from hybrid cells produced by fusing the cells that provide the foreign chromosome or a fragment thereof with the microcells to form cells with high energy. 4. The method according to item 3 of the scope of the patent application, wherein the microcells containing foreign chromosomes or fragments thereof are derived from cells produced by further fusing the microcells derived from the above-mentioned hybrid cells with cells with high microcell formation capacity. 5 · = The method according to item 3 or 4 of the scope of the patent application, wherein the microcell-forming cells are mouse A9 cells. The method according to item 1 of the scope of patent application, wherein the foreign chromosome or a fragment thereof is derived from human normal diploid cells. 7. The method according to item 丨 of the scope of patent application, wherein the cells with multipotential potential are ES cells. 8. The method according to item 丨 of the scope of patent application, wherein the cell with multipotential potential is a disrupted gene homologous to a human antibody. 9. The method according to item 8 of the scope of patent application, wherein the paper standard of the human antibody is compatible with the national standard (CNS) M specification (> &lt; Tear Love) Order Lu Line Due to the destruction of the homologous gene, it was achieved by the target gene homologous recombination method. 10. The method according to item 8 of the scope of the patent application, wherein the host embryo for making chimeric mice is a mouse derived from a gene system lacking a homology to the aforementioned human antibody gene. 11 The method according to the scope of application for patent No. The mice of the system of homologous genes of antibody-like genes were made by the homologous recombination method of the target genes. 12. The method according to item i of the scope of the patent application, wherein the chimeric mouse is a person expressing a human antibody gene on the above-mentioned foreign chromosome or a fragment thereof. 13. The method according to item i of the application, wherein the chimeric mouse is capable of transmitting the above-mentioned foreign chromosome or a fragment thereof to the offspring. 14. The method according to item i in the scope of the patent application, wherein the foreign chromosome is human chromosome 2, human chromosome 14, or human chromosome 22. 15. The method according to item 1 of the scope of the patent application, wherein the above chimeric mouse is one in which at least a part of the mouse cells retains a single foreign chromosome or a fragment thereof. 16. The method according to item 丨 of the patent application range, wherein the above-mentioned conjugated mouse is at least a part of a mouse cell which retains a plurality of foreign chromosomes or fragments thereof. 17 · A method for producing a mouse, which comprises obtaining the offspring of a chimeric mouse produced by the method of the first patent application, and which retains an alien chromosome or a fragment thereof containing a region encoding a human corpus callosum gene And can express the human antibody gene. -2- This paper applies the Chinese National Standard (CNS) A4 specification 7210X297 mm. Λ 8 B8 The scope of patent application is recognized according to the method of item η of the patent scope, in which the aforementioned foreign chromosomes are human chromosome 2 and human chromosome 14 Or human chromosome 22. 19. The method according to item i 7 of the scope of the patent application, wherein the mouse is a part of the soil (the mouse cell retains a single foreign chromosome or a fragment thereof.). According to the application: green patent scope i 7 The method of claim 1, wherein the above-mentioned mouse is a plurality of foreign chromosomes or fragments thereof maintained in at least one mouse cell. According to the method of the “Scope of Patent Application”, the above-mentioned mouse is a mouse carcass gene At least one of them is missing. A method for manufacturing a human antibody is characterized in that: a single or a plurality of human antibody groups with a test base length of 1 Mb or more, 2 foreign wood color m or fragments thereof are stored ^ at least-part of the host cell An immune response in the mouse; and recovering a human antibody against the desired antigen from the mouse. A method for producing a human antibody, characterized in that at least one of the human antibody genes with an age-based length of 1 Mb or more; at least a part of In a host cell; immunizing a desired antigen in at least a human antibody; and 4 ^^ recovered from the mouse against the desired &lt; ^ primitive &gt; Antibody-Standard (CNS) A4gTiI ^ Equipment η Luline-3-1223585 Λ8 B8 C8 ---- D8 VI. Application # s— 24. According to the method of manufacturing a human antibody according to the 22 or 23 of the scope of the patent application, 'Human antibody class or subclass It is selected from the group consisting of IgM, igG, kA, IgD, and their subclasses, and combinations thereof. 25. A method for manufacturing a human antibody according to item 22 or 23 of the scope of the patent application, wherein the human antibody gene is composed of a human heavy chain gene, a human light key kappa gene, a human light chain lambda gene, and a combination thereof Selected in the group. 26. The method of manufacturing a human antibody according to item 22 or 23 of the scope of the patent application, wherein the mouse is a human antibody heavy chain gene region that is retained in at least a part of the mouse cell. 27. The method for manufacturing a human antibody according to item 22 or 23 of the scope of the patent application, wherein the above-mentioned mouse retains all of the human antibody light chain kappa gene region in at least a part of the mouse cell. 28. The method for manufacturing a human antibody according to item 22 or 23 of the scope of the patent application, wherein the above-mentioned mouse is in at least a part of the mouse cell and retains the entire human antibody light chain λ gene region. 29. The method for manufacturing a human antibody according to item 22 or 23 of the scope of the patent application, wherein the mouse is lacking a mouse antibody gene that is homologous to the human antibody gene. 30. The method for manufacturing human antibodies according to item 29 of the patent application, wherein the lack of the stingy antibody gene is achieved by using the target gene homologous recombination method 'by destroying the mouse antibody gene. M. According to the manufacturer of the human antibody according to item 22 or item 23 of the scope of the patent application, it is derived from the B cell of the mouse or the B cell and bone marrow. Standard (CNS) 8-4 specification (21〇χ 297 public love) &quot;-^ '1223585 A8 B8 C8 -----_____ D8 VI. Patent application scope Recovered human antibodies from fusion tumors obtained by cell tumor cell fusion. 32. The method for producing a human antibody according to item 22 or item 23 of the scope of the patent application, which includes a fusion cell obtained from the above-mentioned mouse B cells or fusion of the B cells with bone and bone remains and cell tumor cells Steps for isolating human antibody genes. 33. The method for producing a human antibody according to item 32 of the scope of the patent application, wherein the above-mentioned isolation of the human antibody gene is the isolation of cDNA. 34. The method for manufacturing a human antibody according to item 32 of the scope of patent application, wherein the progress includes the following steps: introducing the isolated human antibody gene into a Shin King cell, and then performing the cell under conditions that can express the human antibody gene Culture and recover the expressed human antibodies. 35. The method for producing a human antibody according to item 34 of the scope of the patent application, wherein the host cell line is selected from CHO cells, BHK cells, liver cancer cells, and myeloma cell. 36 · A method for producing human antibodies, which is to immunize mice with a desired antigen to produce human antibodies against the desired antigens, wherein the mice are retained in at least a portion of mouse cells A foreign chromosome or dragon selected from the group consisting of a human antibody heavy chain gene of 1Mb or more, a human antibody light chain κ gene of iMb or more, and a human light chain λ gene of 1 μb or more, By. Fragment 37. The method for manufacturing a human antibody according to item 36 of the patent application, wherein the antibody is a single antibody. /, 38. The method for manufacturing a human antibody according to item 36 of the patent application, which is -5- Paper Size® S Standard (CNS) Α4 size (210X297 male ^ 7 1223585 A8 : The fusion of B cells and myeloid tumor cells from the above-mentioned mice is used to prepare fusion tumors, and the fusion tumors are cultured to recover human antibodies. According to the method for manufacturing human antibodies according to Item 36 of the scope of the patent application, the fusion of B cells and myeloma tumor cells from the above-mentioned mice is used to make a fusion sigma. The human antibody gene is isolated from the fusion tumor, and The anti-cylinder gene is introduced into a host cell, and the host cell is cultured under conditions that can express human antibody genes, and the expressed human antibody is recovered. 40. The method for manufacturing a human antibody according to item 36 of the scope of application for a patent, wherein a human antibody gene is isolated from the mouse B cells described above, the antibody gene is introduced into a host cell, and the host is cultured under conditions that can express the human antibody gene After the cells, the expressed human antibodies were recovered. 41. The method for producing a human antibody according to item 36 of the scope of application for a patent, which is a method for destroying an endogenous antibody gene in a mouse. 42 · A method for producing an antibody having a heavy chain of a human antibody, characterized in that: at least a part of mouse cells that retain a human chromosome fragment containing a region encoding a human antibody gene are immunized with an antigen, Fusion tumors are produced by fusion of B cells and myeloma tumor cells from the above-mentioned mice; the above-mentioned fusion tumors are cultured to recover antibodies; wherein the above-mentioned mice are used to produce human chromosome fragments 14 containing regions encoding human antibody genes Microcells, chimeric mice or their offspring produced by the method of 'fusion with the microcells' and transferring the chromosome fragments into embryonic stem cells (ES cells). -6- This paper size applies to China National Standard (CNS) A4 (210 X 297 mm) 1223585 A 8 B8 C8 D8 VI. Application for a patent 43. A method for manufacturing an antibody with a human antibody heavy chain, characterized in that: Human No. 14 containing a region encoding a human antibody gene will be retained in at least a portion of mouse cells Mice with chromosomal fragments are immunized with antigens; fusion tumors are made by fusion of B cells and myeloma tumor cells from the mice; human antibody genes are isolated from the B cells or the fusion tumors; and the human antibody genes are introduced into the host Cells, and the cells are cultured under conditions that can express human antibody genes; the expressed human antibodies are recovered; wherein the above mice are used to make microcells containing human chromosome fragments of regions 1 to 4 encoding human antibody genes The chimeric mouse or its offspring produced by the method of transferring the chromosome fragment into embryonic stem cells (ES cells) by fusion with the microcell. 44. The method for manufacturing antibodies with human antibody heavy chain according to item 42 or item 43 of the scope of the patent application, which is a mouse whose endogenous antibody gene is disrupted. 0 This paper applies Chinese National Standard (CNS) A4 Specifications (210 X 297 mm)