KR101348852B1 - Mis18α knockout mouse model and producing method thereof - Google Patents

Abstract

The present invention relates to a Mis18α gene knockout mouse and its embryo, and more particularly, a targeting vector comprising a portion of the Mis18α gene and sequences used for constructing a conditional knockout mouse model. Embryonic stem cells generated using a hit vector (Embryonic Stem Cell), Mis18α knockout mouse embryos obtained using the same, methods for producing and using the same. Mis18α plays an important role in chromosome division during embryonic cell division and can be a very important factor in embryonic cell development. Therefore, the Mis18α knockout mouse model of the present invention can be a good experimental model that can reveal the effects of chromosomal cleavage on the living body and the mechanism of cancer development, and can be a target of new drug development. It can be used to identify new proteins involved in the process. In addition, it can be used to secure a number of new genes involved in the formation of the tautomer, and to develop a technique for analyzing diseases that may occur when an abnormality occurs in the tether formation.

Description

Mis18α knockout mouse model and producing method

The present invention relates to a Mis18α gene knockout mouse and its embryo, and more particularly, a targeting vector comprising a portion of the Mis18α gene and sequences used for constructing a conditional knockout mouse model. Embryonic stem cells generated using a hit vector (Embryonic Stem Cell), Mis18α knockout mouse embryos obtained using the same, methods for producing and using the same.

During cell division, it is very important to replicate the genetic information stored by the cells twice without any error and to accurately transfer the cloned genetic information to each daughter cell through mitosis process. Errors in these processes can cause problems with cell homeostasis and can cause a variety of diseases. Mitosis is divided into prophase, metaphase, telophase, and anaphase. In the middle stage, the spindle is attached to the centromere located in the center of the chromosome, and the tension is formed by the spindle. All chromosomes are arranged on the equator plane of the cell. At this time, a mitotic checkpoint process is performed to check whether all the chromosomes are ready for cleavage without abnormality. Abnormalities in the formation of the chromosome cause abnormal chromosomal division during mitosis because the kinetic core is not attached to the chromosome and there is no connection point of the spindle.

Thus, abnormalities in the formation of centrioles result in different kinds of diseases. In particular, it is well known that patient serum of autoimmune diseases recognizes isotopic proteins, suggesting a link between isotopic and autoimmune diseases. In addition, abnormalities of mitosis during cell division, in particular, result in anemia of chromosome, and abnormalities of chromosome commonly found in cancer cells may be one of the major causes of cancer development or progression. Claims have been identified in studies using genetically engineered mice that reduced the expression levels of several proteins responsible for mitotic checkpoints (Holland AJ, Cleveland DW. Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis. Nat Rev Mol Cell Biol . 2009 Jul; 10 (7): 478-487).

The types and mechanisms of the proteins responsible for the mitosis checkpoint process have been in-depth, and as a result, the role of checkpoint proteins in the mid to late mitosis process has been studied in detail. In addition, in studies using a gene-deficient mouse model, a decrease in the amount of expression of the proteins constituting the checkpoint (e.g. Mad1, Mad2, Bub1, Bub3, BubR1) results in abnormal chromosome numbers and in the long term. Has been shown to promote cancer development (Holland and Cleveland, 2009). Many of the proteins in the isotope are selectively loaded and separated during the mitosis process, so they may be directly related to mitosis and checkpoint regulation, but little has been studied about the functional network of mitogens.

The centromere site is the site where kinetochore attaches during chromosome cleavage and is an essential site in both mitosis and meiosis. Attention (Holland and Cleveland, 2009). In yeasts where most of the studies on isotope abnormalities have been studied, the gene sequence has been studied as an important factor determining the site of the centrosome, but in mammals, it has been found that the gene sequence does not play an important role in forming a centromere.

In humans, a repetitive sequence of DNA called alpha satellite DNA was thought to act primarily as a site for forming a centromere.However, if a centrosome is missing from a particular chromosome, another site on the chromosome might act as a new centromere. It turned out. It has been observed that if the isotopes are formed at a second position other than the original one, they could be formed at a sequence other than the alpha satellite DNA sequence. Since the discovery of the first neocentromere in 1993, to date, more than 90 neocontromere have been found in human chromosomes, with new ones being formed at positions other than the original ones. Thus, something other than the DNA sequence was thought to designate the isotopic site (Marshall OJ et al., Am J Hum Genet . 2008 Feb; 82 (2): 261-282). Until recently, the most widely accepted papers have suggested that Cenp-A, a similar protein of histone protein H3, designates a conformation site rather than a specific DNA sequence (Zeitlin SG. Epigenetics . 2010 Jan 1; 5). 1): 34-40).

That is, the general nucleosome is composed of octamers containing H2A, H2B, H3, and H4, whereas the nucleosomes of the isotope region contain Cenp-A instead of histone H3, and thus the octamers containing H2A, H2B, Cenp-A, H4, or It has the structure of tetramer. Recent studies have shown that isotope positioning by the CENP-A protein can be controlled in a variety of epigenetic ways. Therefore, CENP-A is found to be another regulator that brings chromosomal abnormalities when its function is abnormal, and is thought to be a target protein of diseases occurring by chromosomal abnormalities. It has been found that Cenp-A plays an important role in designating a conformation site, but until recently, how newly synthesized Cenp-A finds a conformation site, which proteins are involved in this process, and the role of each protein Nothing has been studied about what.

As a prior document related to the present invention, Fujita Y et al., Published in 2007, Priming of centromere for CENP-A recruitment by human hMis18α, hMis18β, and M18BP1. Dev Cell . 2007 Jan; 12 (1): 17-30, for the first time, found that Cenp-A did not migrate to the site of the centromere without the Mis18α and Mis18β proteins. However, this did not search for the function of the Mis18α gene using the conditional knockout animal model of Mis18α as in the present invention, and also did not elucidate the function of Mis18α.

In Korea, many studies have been conducted on cell cycle interphase and interstitial checkpoint regulation. However, only a few researchers have been concerned about mitosis and mitotic checkpoint regulation. And studies on the linkage with human diseases related to the lack of separation have not been reported in Korea or abroad. Conjugate formation and segregation may be directly related to mitosis and checkpoint regulation, but no functional network of mitogens by mitosis has been studied systematically. Until now, functional studies of allogeneic proteins have been studied at the yeast or cell line level. Most of what went on in

Therefore, the present inventors have revealed the role and mechanism of action of Mis18α and Mis18α binding proteins in the process of centromere formation by Cenp-A, and the Mis18α knockout animal model for the purpose of elucidating the developmental and pathological significance of the formation of the centromere. During the study of the present invention was completed.

Accordingly, an object of the present invention is to provide a targeting vector capable of knocking out a mouse Mis18α gene.

Another object of the present invention is to provide an embryonic stem cell of a mouse into which a mouse Mis18α gene targeting vector is introduced.

In addition, another object of the present invention is to provide a chimera mouse using embryonic stem cells into which the hit vector is introduced.

It is another object of the present invention is to provide a Mis18 α ^{f / +} transgenic mice are Mis18α one gene targeting.

Another object of the present invention is to provide a Mis18α ^{+ / Δ} transgenic mouse from which one gene of Mis18α has been removed.

In addition, another object of the present invention to provide a method for producing the Mis18α ^{+ / Δ} transgenic mice.

In addition, another object of the present invention Mis18α ^{Δ / Δ} knocked out Mis18α gene To provide a transgenic mouse embryo.

In addition, another object of the present invention is to provide a method for producing the Mis18α ^{Δ / Δ} transformed mouse and Mis18α ^{Δ / Δ} transformed mouse embryo.

Another object of the present invention is to provide a method of using a Mis18α ^{+ / Δ} transgenic mouse lacking the Mis18α gene as a disease animal model associated with abnormal cell division.

Another object of the present invention is to provide a method for screening a disease therapeutic agent and a prophylactic agent associated with cell division disorders using the Mis18α ^{Δ / Δ} transgenic mouse embryo.

In order to achieve the object of the present invention as described above, the present invention is from the LoxP (locus of X-over P1) site, the second Eco RI cleavage site in the 5 'direction of the first exon of the mouse Mis18a genomic gene. A mouse Mis18α gene targeting vector is provided comprising a DNA sequence consisting of a gene segment up to Exon 2, a neomycin resistance gene, and an Eco RI cleavage site.

In one embodiment of the present invention, the neomycin resistance gene may be formed in the order of the Flip Recognition Target (FRT) site, LoxP site, neomycin resistance gene, FRT site and LoxP site.

In addition, the present invention provides a cell line of a mouse in which the Mis18α gene targeting vector is introduced and homologous recombinated.

In addition, the present invention provides embryonic stem cells of mice in which homologous recombination is introduced by introducing a Mis18α gene targeting vector.

The present invention also provides a chimera mouse prepared by injecting the embryonic stem cells into the blastocyst of a normal mouse and implanting the embryonic stem cells into a surrogate mother.

In addition, the present invention is Mis18α one gene having a Mis18 α ^{f / +} genotype produced by mating the chimeric mouse and the normal mouse provides phlox de transgenic mice.

The present invention also provides the α Mis18 ^{f / +} genotype mice, with Cre Mis18α one gene deficient mice with Mis18 α ^{+ / Δ} genotype prepared by mating the mice that express the recombinase.

The present invention also provides a gene-deficient mouse embryo Mis18α having Mis18 ^Δ α ^{/ Δ} genotype that is produced by mating the Mis18α one gene deficient mice.

In addition, the present invention comprises the steps of preparing a mouse Mis18α gene hit vector; Introducing the targeting vector into embryonic stem cells to obtain a Mis18α gene-targeting embryonic stem cell clone by homologous recombination; Injecting the Mis18α gene-targeted embryonic stem cells into the inner cell mass of the blastocyte of a normal mouse and implanting the same into a surrogate mother mouse to obtain a chimera mouse; Obtaining Mis18α one gene targeting mouse having Mis18 α ^{f / +} genotype via mating from the chimeric mice, by mating the Mis18 α ^{f / +} genotype mice, and Protamine-Cre mice Mis18α one gene having the Mis18 α ^{+ / Δ} genotype Provided is a Mis18 α ^{+ / Δ} transgenic mouse comprising obtaining a deficient mouse.

In one embodiment of the present invention, the mouse Mis18α gene targeting vector may be the Mis18α gene targeting vector of the present invention described above.

In one embodiment of the present invention, in the case of the step for obtaining Mis18α one gene deficient mice with Mis18 α ^{+ / Δ} genotype via mating from the chimeric mice from the method, to the chimeric mice bred with normal mice Mis18 α obtaining ^{f / +} mice; Mating Mis18 α ^{f / +} mice with mice expressing Flp recombinant enzyme (Flippase); Selecting mice expressing the Flp recombinase from among the progeny obtained from the cross and at the same time showing the Mix18α one-side gene floxed (f / +) genotype; Mating the selected mice with normal mice to obtain a mouse from which a neomycin resistance gene has been removed; Mating the mouse from which the neomycin resistance gene has been removed with a mouse that expresses Cre recombinase; Selecting mice expressing Cre recombinase at the same time of Mis18α gene phloxed (f / +) genotype among progeny obtained through the crossing; And mating the selected mice with normal mice to obtain mice in which exons 1 and 2 are removed from one gene of Mis18α.

The present invention also provides a method of using a Mis18 α ^{+ / Δ} Transgenic Mice with a genetic deficiency Mis18α one as an animal model of diseases associated with abnormal cell proliferation.

In addition, the present invention Mis18 α ^{Δ / Δ} knocked out of the Mis 18α gene In transgenic mouse embryos, the embryos may exhibit characteristics that do not develop into individuals and die due to chromosomal division abnormalities during cell division.

In addition, the present invention comprises the steps of treating the Mis18 α ^{/ Δ} embryo with a disease treatment and prevention agent candidates associated with cell division abnormalities; And comparing the cell division process of the untreated Mis18 α ^{Δ / Δ} embryo with the therapeutic substance and selecting a substance that has restored cell division ability. It provides a screening method.

In one embodiment of the present invention, the disease may be cancer or autoimmune disease.

In the dividing cells of the Mis18α knockout mouse embryo of the present invention, cell division did not proceed normally, such as chromosomes not being properly arranged, and the embryos eventually died. This suggests that Mis18α plays an important role in chromosome division during embryonic cell division and is a very important factor in embryonic cell development. Therefore, the Mis18α knockout mouse model of the present invention can be a good experimental model that can reveal the effects of chromosomal cleavage on the living body and the mechanism of cancer development, and can be a target of new drug development. It can be used to identify new proteins involved in the process. In addition, it can be used to secure a number of new genes involved in the formation of the tautomer, and to develop a technique for analyzing diseases that may occur when an abnormality occurs in the tether formation.

Figure 1 is a figure showing the identification and expression of Mis18α (NAP1), (A) When NcoR and Mis18α together in the yeast expression can be confirmed whether the binding of each other through the increase in the expression of the reporter β-gal. (B) Mis18α protein is composed of 204 amino acids and zinc finger and coiled-coil domain. (C) DNA sequence and amino acid sequence of Mis18α. (D) Photograph showing the results of the expression of Mis18α mRNA after purifying RNA from mouse tissues. (E) Mis18α and NcoR confirmed the intracellular binding.
2A and 2B analyze the characteristics of Mis18α (NAP1). FIG. 2A is a result of examining the binding of various NcoR deletion mutants and GST-Mis18α to determine where Mis18α binds to NcoR. 2B shows experimental results for examining the function of Mis18α in relation to transcription.
3A and 3B are photographs showing the results of in situ hybridization in order to analyze the expression patterns of Mis18α mRNA according to developmental stages in detail. FIG. 3A is an embryo and FIG. 3B is collected after birth. .
4 shows the expression of Mis18α in adult mice.
5A and 5B are photographs showing the PEST sequence and intracellular stability on Mis18β (OIP5), and FIG. 5A shows the proteins degraded by the ubiquitin-proteasome system intracellularly at the N-terminal site of Mis18β (OIP5). Figure 5B is a diagram showing the presence of a large number of PEST sequences found, Figure 5B is an illustration of the expression of the independent expression of Mis18β (OIP5) in the cell and when expressed with Mis18α expression changes.
6 is a diagram illustrating a method of obtaining a Mis18α gene hit vector and a Mis18α gene deficient mouse according to an embodiment of the present invention from a genetic perspective.
7 is a result of Southern blotting to verify the homologous recombination of the hit vector introduced into embryonic stem cells after preparation of the Mis18α gene floxed embryonic stem cells of the present invention.
8 to 11 show the effects of the Mis18α gene defect on the early embryonic stage. FIG. 8 shows normal mice and mice lacking both Mis18α genes of mice in which one gene of Mis18α was removed from the genome. Mis18 α ^{Δ / Δ} ) is a table showing the genotype of each stage of embryonic formation.
9 shows Mis18 α ^{+ / Δ} This is the result of analyzing genotypes of pups when a female (79 mice) is obtained by crossing a male and female genotype.
10 shows Mis18 α ^{Δ / Δ} This study confirmed the survival of 3.5 days of pregnancy in genotyped mouse embryos.
FIG. 11 shows the growth of the embryos extracted at 3.5 days of gestation and then artificially cultivated after the mice with one side of the Mis18α gene were crossed to each other in order to analyze the embryo's death time more precisely.
12A to 15 are the results of analyzing the effect of Mis18α gene deletion on chromosome division and apoptosis. FIG. 12A shows chromosomes and spindles in embryos of normal mice. FIG. 12B is abnormal in Mis18α gene deficient mouse embryos. The photo shows the chromosomes and spindles arranged.
Figure 13A is a photograph showing the cell division process in the inner ear of normal mice.
Figure 13B is a photograph showing the cell division process in Mis18α gene deficient mouse embryo.
Figure 14 is a photograph showing the distribution pattern in the nucleus of Mis18α gene deficient mouse embryo and normal mouse embryo of CENP-A protein located only in the centromere.
Figure 15 shows the results of apoptosis difference between the Mis18α gene deficient mouse embryo and normal mouse embryo cells by the TUNEL method.

The present invention was the first to provide a Mis18α gene knockout animal model and Mis18α gene knockout animal embryos, and to provide the first and the same effects on cell association and cell death including the formation of Mis18α. It is characterized by the point.

In one embodiment of the present invention, the Mis18α gene knockout mouse embryo of the present invention has been found to be abnormally aligned with chromosomes and spindles relatively relatively (FIG. 12), and when chromosomes are not properly arranged in dividing cells. The chromosomes that were cut off and separated were observed (FIG. 13), and the spindle was not attached to the chromosome, or there was a problem that the chromosomes were gathered around the mitosis before the chromosome was cut at the end of chromosome division. The phenomenon was found. This implies that Mis18α plays an important role in chromosome division during embryonic cell division and is particularly important for embryonic cell development.

In addition, the effect of the Mis18α gene on the synthesis of the histone H3 analogue of CENP-A, which is an analog of histone H3, was tested. As a result, CENP-A stained strongly in most cell nuclei in normal embryonic cells. However, the cells of the mouse embryo of the Mis18α gene deficient mice did not show a spot staining in the nucleus (FIG. 14), and consequently, the deletion of the Mis18α gene prevented the CENP-A from being located in the centromere. This results in abnormal chromosome division in cell division and eventually leads to cell death. As a result, embryos lacking the Mis18α gene could no longer develop into individuals due to apoptosis.

Therefore, the Mis18α knockout animal model of the present invention can be used as an animal model for identifying mechanisms related to abnormal cell division processes including mobilized or abnormal chromosomal division, or screening prophylactic or therapeutic agents associated with diseases. .

However, as described above, the Mis18α gene, which is the subject of the present invention, plays a very important role in the embryonic development stage. Thus, when preparing a knockout animal model according to the existing method, normal embryo growth and As it is impossible to generate an organism, it is possible to manufacture an embryo, but as a result, an individual having a gene knockout genome cannot be obtained. Therefore, it is impossible to accurately analyze which mechanism plays a specific role in vivo.

Accordingly, the present inventors have devised a conditional knockout mouse model according to the present invention to compensate for the above problems. This method constructs mice with specific loxP sequences located on both sides of the important exon region of the gene to be removed, and crosses them with mice expressing a recombinant enzyme called Cre to remove exons between loxPs by the action of Cre enzyme. Induce knockout of specific genes. The advantage of this method of manufacturing a conditional knockout animal model is that if a specific gene plays an important role in the developmental stage and a general knockout animal model cannot be obtained, mice that express Cre only in specific tissues (cross) You can get mice (animals) that have been knocked out genes selectively in only certain tissues, not whole tissues. In other words, the conditional knockout animal model according to the present invention induces limited knockout of genes in selective and conditional individuals through crosses with various tissue-specific or drug-induced Cre-expressing mice. There is a big advantage to be able to study the physiological function in vivo completely.

The Mis18α knockout animal model according to the present invention may be manufactured using a mammal except a human, and the mammal except the human may be a monkey, a rat, a mouse, a rabbit, a dog, a primate, and the like, but is not limited thereto. Do not. Preferably it may be a Murida animal. In addition, the Mis18α gene according to the present invention is a gene having a cDNA nucleotide sequence of SEQ ID NO: 1, Mis18α protein encoded from the Mis18α gene is a protein having an amino acid sequence of SEQ ID NO: 2.

Mis18α four according to the present invention out animal models LoxP (locus of X-over P1 ) region, mice Mis18α genomic gene 1 from the second nearest Eco RI cleavage sites in 5 'direction of the second exon (exon) to the second exon A mouse Mis18α gene targeting vector comprising a DNA sequence consisting of a gene fragment, a neomycin resistance gene and an Eco RI cleavage site may be prepared.

The neomycin resistance gene, preferably, may be made in the order of a Flip Recognition Target (FRT) site, LoxP site, neomycin resistance gene, FRT site and LoxP site.

Although not limited thereto, the Mis18α gene targeting vector may be a vector having a nucleotide sequence of SEQ ID NO: 3 or having a configuration of the hit vector shown in FIG. 6.

In one embodiment of the present invention Mis18 α ^{Δ / Δ} knocked out Mis18α gene of the present invention Transgenic mouse embryos can be prepared by a method comprising the following steps.

(a) obtaining a Mis18α genomic DNA clone comprising the Mis18α gene; (b) preparing a Mis18α conditional knockout cassette using the Mis18α genomic DNA clone and known sequences; (c) transfecting the conditional knockout cassette into embryonic stem cells; (d) injecting the embryonic stem cells into a blastocyst; (e) implanting the embryo into which the embryonic stem cells have been injected into the uterus of the surrogate mother to obtain a chimeric mouse; (f) crossing the chimeric mice to obtain heterozygous F1 transgenic mice having a Mis18 α ^{f / +} genotype; (g) Mis18 α ^{f / +} mice, the mice exhibit Flp recombinase (Flippase) while expressing the Flp recombinase from the offspring obtained by mating the mice that express at the same time Mis18α one gene phlox de (floxed) to (f / +) genotype Screening; (h) crossing the selected mice with normal mice to obtain a mouse from which a neomycin resistance gene has been removed; (i) Selecting mice expressing Cre recombinase at the same time as having a Mis18 α gene phloxed (f / +) genotype among progeny obtained by crossing the mice with the neomycin resistance gene removed with mice expressing Cre recombinase Doing; (j) Mis18 α ^{+ / Δ} from which exons 1 and 2 were removed from one gene of Mis18α by crossing the selected mice with normal mice. Obtaining a mouse having a genotype; (k) Mis18 α ^{+ / Δ} Mice were crossed to Mis18 α ^{Δ / Δ} The step is to obtain homozygous transgenic mouse embryos having the genotype.

In the step (a), the Mis18α genomic DNA clone is purified from target cloning vectors such as chemical synthesis, PCR amplification, eukaryotic cells or plasmids containing the target nucleotide sequence, phagemids, YACs, cosmids, and bacteriophages. It can be obtained by a method known in the art such as. In one embodiment of the present invention, Mis18α gene fragments could be obtained by PCR amplification using primers that specifically recognize Mis18α genes in mice.

Conditional knockout of the Mis18α gene in step (b) can be generated by inserting the loxP sequence on both sides of the Mis18α exon to be removed. The insertion site is an intron site so as not to interfere with the expression of the native gene. The present inventors can induce the deletion of the Mis18α gene by homologous recombination by introducing the Mis18α conditional knockout cassette prepared above into embryonic stem cells.

Embryonic stem cells (ES cells) of step (c) can generally be obtained from pre-implantation embryos cultured in vitro (Evans et al., Nature, 292). : 154-156, 1981; Bradley, et al., Nature, 309: 225-258, 1984; Gossler et al., Proc. Natl. Acad. Sci USA, 83: 9065-9069, 1986). The ES cells can be cultured using methods known in the art (Robertson, "Teratocarcinomas and Embryonic Stem Cells, a Practical Approach", IRL Press, Washington DC, 1987; Bradley et al., Current Topics in Devel. Biol. 20: 357-371, 1986; Hogan et al., "Manipulating the Mouse Embryo": A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).

Introduction of the Mis18α knockout cassette into embryonic stem cells may be performed by methods known in the art. For example, pronuclear microinjection, retrovirus mediated gene transfer, gene targeting, electroporation, seminal mediated gene transfer, calcium phosphate / DNA co-precipitation and Microinjection and the like. In one embodiment of the present invention, Mis18α knockout cassette was introduced into embryonic stem cells by electroshock. After transfecting embryonic stem cells, only those cells which have been homozygous are selected by selecting embryonic stem cell clones which are resistant by culturing in a selection medium containing a specific antibiotic.

In step (d), the embryonic stem cells of step (c) are injected into the blastocyst of the cyst. In step (e), the embryos injected with the embryonic stem cells are transplanted into the uterus of the surrogate mother to obtain a chimeric mouse.

In step (f), a heterozygous F1 transgenic mouse having Mis18 α ^{f / +} genotype is induced from the chimeric mouse, and in step (g), the Mis18 α ^{f / +} mouse expresses Flp recombinant enzyme (Flippase). Mice that express the Flp recombinase and express the Mix18α gene phloxed (f / +) genotype among the progeny obtained from the mating mice were selected, and the mice selected in step (h) were crossed with normal mice by neomycin. The phlox mice were obtained with one Mis18α gene from which the resistance gene was removed.

In step (i), the Mis18α single gene phloxed mouse from which the neomycin resistance gene has been removed is crossed with a Cre recombinant enzyme-expressing mouse, which has a Mis18 α single gene phloxed (f / +) genotype and simultaneously Cre recombination. Enzyme-expressing mice were screened and mice selected in step (j) were crossed with normal mice to remove Mis18 α ^{+ / Δ} exons 1 and 2 from one gene of Mis18α. Mice with genotypes are obtained.

In the step (k), Mis18 α ^{+ / Δ} mice are crossed to each other to Mis18 α ^{Δ / Δ} Although homozygous transgenic mouse embryos having genotypes can be obtained, other methods of germline transmission may be used depending on the type of knockout cassette used.

According to one embodiment of the invention Mis18 α ^{Δ / Δ} knocked out of the Mis18α gene Transgenic mouse embryos are characterized by dying, failing to develop into normal individuals due to abnormal chromosomal division during cell division.

Therefore, the present invention provides a method for manufacturing a Mis18α gene deficient mouse, comprising the steps of preparing a mouse Mis18α gene targeting vector capable of selectively removing exons 1 and 2 of the Mis18α gene of the mouse; Introducing the targeting vector into embryonic stem cells to obtain an embryonic stem cell clone in which a part of the Mis18α gene has been removed by homologous recombination; Injecting the Mis18α gene deficient embryonic stem cells into the inner cell mass of the blastocyte of a normal mouse and implanting the same into a surrogate mother mouse to obtain a chimera mouse; And it provides a step for obtaining the rat Mis18α one gene is removed with Mis18 α ^{+ / Δ} genotype via mating from the chimeric mice.

According to an embodiment of the present invention, when the mouse Mis18α gene hit vector includes a FRT (Flippase Recognition Target) site and a LoxP site, the method of obtaining a mouse from which the Mis18α one gene is removed from the chimeric mouse in the method may include: Mating mice with mice expressing Flp recombinant enzyme (Flippase); Selecting mice expressing the Flp recombinase from among the progeny obtained from the cross and at the same time showing the Mix18α one-side gene floxed (f / +) genotype; Mating the selected mice with normal mice to obtain a mouse from which a neomycin resistance gene has been removed; Mating the mouse from which the neomycin resistance gene has been removed with a mouse that expresses Cre recombinase; Selecting mice expressing Cre recombinase at the same time of Mis18α gene phloxed (f / +) genotype among progeny obtained through the crossing; And mating the selected mice with normal mice to obtain mice in which exons 1 and 2 are removed from one gene of Mis18α.

The term "flocxed" refers to a DNA sequence state sandwiched between two LoxP sites in genetics, and is abbreviated as "flanked by LoxP." Recombination between LoxP sites is catalyzed by Cre recombinase. Gene fluxing allows knocked out, translocated, or inverted genes, and the phloxed genes are for organ-specific knockouts purposes. Used.

In addition, the "recombinase" is also called "Cre", a type I topoisomerase derived from P1 bacteriophage, and site-specific DNA recombination between LoxP sites Catalyzes. Cre recombinase is useful as a tool for modifying genes and chromosomes and is used to remove DNA segments (called 'floxed') surrounded by LoxP sites in experimental animals.

Therefore, the present invention can produce a Mis18α gene deficient mouse through the above method, the mouse Mis18α gene targeting vector is introduced into the embryonic hepatocytes or mouse cell line of the homologous recombination mouse, the embryonic stem cells of the mouse to the microbial bag of normal mice Chimeric mice prepared by injection and implantation in surrogate mothers can be provided.

Further, with the chimeric a mice prepared by mating with normal mouse one gene phlox de (f / +), as well as Mis18 α ^{f / +} Mis18α one gene targeting mouse of the genotype with the genotypes, the Mis18 α ^{f / +} genotype mice Mice expressing Cre recombinase can be crossed to provide transgenic mice of the Mis18 α ^{+ / Δ} genotype. The mouse expressing the Cre recombinase may be a Protamine-Cre mouse having a Cre recombinase, whose expression is controlled by a protamine promoter. In addition, it is possible to provide a gene-deficient mouse embryo Mis18α having Mis18 α ^{+ / Δ} genotype produced by mating the Mis18α one gene deficient mice.

Meanwhile, the present invention can be used in an in vitro assay for screening a therapeutic or prophylactic agent for a disease associated with abnormalities of cell division processes including abnormalities of mobilization or chromosomal division.

Although not limited thereto, the disease may be a cancer or an autoimmune disease that is well known in the art due to abnormal chromosome division and abnormal cell division.

The therapeutic agent may be not only to increase the expression of Mis18α but also to inhibit the function of Mis18α or reduce the expression of Mis18α in some cases.

More specifically, the analysis method of the present invention comprises the steps of: (a) observing the cell division process according to the initial time of division of Mis18 α ^{Δ / Δ} knockout mouse embryos and Mis18 α ^{+ / +} wild type mouse embryos; (b) administering a therapeutic agent to the Mis18 α ^{Δ / Δ} mouse embryos and the Mis18 α ^{+ / +} wild type mouse embryos and observing cell division patterns for the same time; And (d) to Mis18 α ^{+ / +} wild-type cell division process Mis18 α ^{Δ / Δ} transgenic mouse embryo compared to the progress promote rate in mice embryos comparing a change degree of cell division pattern following administration prior to administration of the therapeutic substance Selecting a substance that increases the rate of promotion in the cell.

As used herein, "therapeutic or prophylactic agent" includes any substance, molecule, element, compound, entity, or combination thereof. But are not limited to, proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, and the like. It may also be a natural product, a synthetic compound or chemical compound, or a combination of two or more substances.

The present invention also provides an in vivo analysis method for screening a therapeutic or prophylactic agent for a disease associated with abnormalities of cell division processes including abnormalities of mobilization or chromosomal division. The substance may be to promote the function of Mis18α or to increase the expression of Mis18α in the subject.

More specifically, the assay method of the present invention comprises the steps of (a) preparing a DNA construct comprising a nucleic acid encoding a Mis18α gene and / or a promoter of the Mis18α gene operably linked to a reporter gene; (b) contacting the DNA construct with a therapeutic agent; And (c) selecting an agent that inhibits expression of the reporter gene by measuring the expression level of the reporter gene.

The term "reporter gene" refers to a gene used to easily observe the expression of a gene of interest with high sensitivity. More specifically, the reporter gene is used in place of directly detecting the expression of the target gene by adjusting its expression or production according to the expression level of the target gene, and directly or indirectly, its activity or There is a characteristic that can detect the degree of production. Such genes are known in the art and include, but are not limited to, for example, chloramphenicol acetyl transferase (CAT), β-galactosidase and luciferase. Methods of measuring their activity or degree of production are also known in the art.

As used herein, "DNA construct" refers to a DNA fragment artificially assembled to be transformed into a target tissue, cell line or animal. Preferably, the DNA construct comprises a target gene sequence, a regulatory sequence and a reporter gene sequence. By "operably linked" above is meant that the appropriate nucleic acid is linked in such a way as to enable gene expression when bound to expression control sequences.

Thus, the increased expression of the reporter gene due to the presence of the therapeutic agent means that the candidate may be useful for causing normal cell division by increasing Mis18α expression.

Thus the invention can provide a switching Mis18α one gene is transfected deficient Mis18 α ^{+ / Δ} animal and a method of using as a cell line, using the animal embryo, an animal model of diseases associated with abnormal cell division, cell line experiments.

Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

≪ Example 1 >

Mis18 alpha ( NAP1 ) Identification and Characterization of Proteins

The inventors discovered a new ORF by the yeast two hybrid screening method using the inhibitor domain of NcoR as bait and named it NcoR-Associated Protein (NAP) 1. It was identified as a new gene with unknown function, but was reported under the name Mis18α during the study. The present inventors intend to follow the name Mis18α.

Mis18α is a protein consisting of 204 amino acids and has a zinc finger and a coiled-coil domain (FIG. 1B). The DNA sequence and amino acid sequence of Mis18α are represented by SEQ ID NO: 1 and SEQ ID NO: See Figure 2.

After purifying RNA in each tissue of the mouse, the expression of Mis18α mRNA was observed. As a result, the testis had the highest expression, and it was confirmed that it was also expressed in the spleen and lung (FIG. 1D).

In order to analyze the characteristics of the Mis18α gene, when NcoR and Mis18α were expressed together in the yeast, as shown in FIG. 1A, it was confirmed that the binding of the reporter gene β-gal was increased.

In addition, in order to confirm whether Mis18α and NcoR bind intracellularly, Myc-Mis18α was expressed in HeLa cell line, followed by immunoprecipitation with an antibody against NcoR, followed by immunostaining using an antibody against Myc-tag. As shown in E of FIG. 1, it was confirmed that NcoR and Mis18α bind intracellularly.

Next, the present inventors examined the binding of various NcoR deletion mutants and GST-Mis18α to determine where Mis18α binds to NcoR. As a result, as shown in FIG. 2A, it was found that Mis18α binds to the site including the repressor domain I of NcoR. In addition, in order to examine the function of Mis18α in relation to transcription, it was found that Mis18α has a function of inhibiting the transcription of β-gal reporter by connecting Mis18α to the DNA binding domain of GAL4. 2B).

< Example  2>

In situ hybridization  Method and adult development of mice Mis18 Expression Pattern Analysis of α

We performed in situ hybridization to analyze the expression pattern of Mis18α in more detail (FIG. 3). Mis18α mRNA shows relatively even expression in tissues throughout the body at embryonic age 9.5 (e9.5) and e12.5, but as development progresses to e15.5 the overall expression decreases and some tissues such as skin, thymus and kidney It shows a specific expression pattern (Fig. 3A). This feature persisted after the offspring were born (FIG. 3B).

In addition, although the expression of Mis18α mRNA in the adult as a whole, the expression level was significantly lower than that of the fetus, and only a few tissues showed high expression of Mis18α (FIG. 4). In the tissues with high expression of Mis18α, the expression of Mis18α was found in the sites where cell divisions are frequently caused by ovaries (particularly oocytes), testes, skin, thymus, spleen, and kidneys. The information on the expression pattern of Mis18α provided important information when deciding which tissue to remove from the tissue when analyzing Mis18α conditional gene deficient mice.

< Example  3>

Mis18 Identification of α-binding proteins

The present inventors performed the following experiment to find a protein that binds to Mis18α. Mis18α having a 3 × flag tag was expressed in HEK293T cell line and cell fractions were obtained. Anti-flag antibody (M2) -agarose is used to pull down the proteins with flag tags (Flag-tag) and then 3x flag peptide (Flag peptide) The binding protein was purified purely. The obtained proteins were identified using mass spectrometry. The same experiment was performed a total of four times to classify the proteins repeatedly identified in all four times as proteins that are likely to bind Mis18α (see Table 1).

Mis18α binding protein Gene Symbol Protein Name CCDC144A coiled-coil domain containing 144A OIP5 Opa interacting protein 5 (Mis18β) YEATS4 YEATS domain containing 4 (GAS41) MLSTD1 Male sterility domain containing 1 MLSTD2 Male sterility domain containing 2

In addition, the present inventors screened a mouse embryonic 17-day-old library using mouse Mis18α as a bait to identify proteins that bind to Mis18α. Activating transcription factor 5 (Atf5), var1,3-like 1 inhibitor (Supv3l1), DNA methyltransferase1 associated protein1 (Dmap1) And four genes were finally selected, including centrosomal protein 27 (Cep27).

OIP5, identified as a protein that binds to Mis18α, has been reported under the name Mis18β because it works with Mis18α during the Cenp-A loading process. The present inventors in the present invention in the process of analyzing the primary structure of OIP5 in the N-terminal region of OIP5 is found in the PEST sequence (sequence) that is found in many proteins that are degraded by the ubiquitin-proteasome system in the cell Was found (FIG. 5A).

In addition, when OIP5 is expressed independently of the cells, it was found that the expression was not good, and when expressed with Mis18α, it was confirmed that the amount of expression was significantly increased (FIG. 5B). From the above results, the inventors found that OIP5 is a very unstable protein, and is an important target for regulating the function of the Mis18 complex.

< Example  4>

Mis18 α Gene Deletion Hit Vector Preparation

The present inventors attempted to design a hit vector to simultaneously remove exon 1 and exon 2, which contain transcription start codons (ATG), among the five exons constituting the Mis18α gene. First, a corresponding region of about 10.5 kb in size was obtained from the genomic DNA of C57BL / 6 strain mice and introduced into the pSP72 vector (promega). The target site containing exons 1 and 2 is about 2.5 kb in size and inserts LoxP sequences at both ends (floxed) to remove exons 1 and 2 by the action of Cre recombinase. It was. A short homology arm of about 1.2 kb was placed on the 3 'side of exon 2, and a long homologous recombination site of about 6.5 kb on the 5' side of exon 1. The neomycin resistance gene (Neo gene) enclosed by the LoxP sequence and the FRT sequence was cut out from pGK-gb2 LoxP / FRT-Neo and placed on 3 'side of exon 2. The total size of the hit vector thus prepared was 14.7 kb, and the nucleotide sequence thereof was as described in SEQ ID NO: 3 (see FIG. 6).

< Example  5>

Mis18 α gene Phlox ( floxed ) Embryonic stem cell  Produce

In order to prepare Mis18α gene fluxed embryonic stem cells, the present inventors cut and linearized 10 μg of the hit vector prepared above with restriction enzyme Cla I, and then introduced the cells into embryonic stem cells of C57BL / 6 mice by electroshock method. Surviving embryonic stem cell clones were treated by G418, and the hit vectors were homologously recombined by Southern blotting.

Embryonic stem cells in which a hit vector has been introduced into genomic DNA will survive in media containing G418 because they contain neomycin resistance genes. In order to verify the homologous recombination of the target vector introduced into the embryonic stem cells, genomic DNA was extracted from each embryonic stem cell, digested with Eco RI restriction enzymes, and Southern blotting using the probe shown in FIG. 1. Was performed.

By including the artificial Eco RI recognition sequence at the 3 'region of the neomycin resistance gene of the vector hits the size difference between the fragments when digested with Eco RI restriction enzyme analysis made it possible to Southern blotting. As a result, the normal Mis18α genomic gene was found to be about 8.4 kb in size by Eco RI restriction enzyme, and when the hit vector was inserted into the genome by homologous recombination, it was about 6.6 kb in size by Eco RI restriction enzyme. (FIG. 7). Therefore, the inventors were able to know that the embryonic stem cells of the present invention introduced a hit vector and homologous recombination occurred.

< Example  6>

Embryonic stem cells  Used Mis18 α gene Phlox  Production of mice (f / +)

The present inventors microinjected the Mis18α gene phlox embryonic stem cells obtained above into the blastocyst inner cell mass of Balb / c mice and implanted them into surrogate mother mice to prepare chimeric mice. Among the chimeric mice, male mice with a high specific gravity of black hair were selected and crossed with C57BL / 6 female mice to produce F1 progeny. The tails of the three-week-old F1 offspring thus produced were cut about 0.5 cm, and genomic DNA was extracted from the fragments. The PCR primers used at this time are as follows.

SDL1: 5’-CTCCGTTCCGTCTTGGCACAC-3 ’(SEQ ID NO: 4),

LOX1: 5'-CCTAAGTCGTTGACCTGACCGAGG-3 '(SEQ ID NO: 5).

For offspring delivered with the hit vector, the LoxP sequence was added, resulting in a 380 bp PCR product, and for the normal Mis18α genomic gene, a 320 bp PCR product.

< Example  7>

Neomycin resistance gene removed Mis18 α gene Phlox  Production of mice (f / +)

When neomycin resistance genes remain in mice, there are known cases of inhibiting the expression of peripheral genes. Thus, the neomycin resistance genes were removed from phlox mice (f / +). Females of one of the Mis18α gene phloxed mice (f / +) and FLPeR mice (The Jackson Laboratory) expressing Flp recombinase were crossed to obtain pups. Among the pups, males expressing the Flp recombinase and simultaneously showing the Mis18α one-sided phlox (f / +) genotype were crossed with females of C57BL / 6 strains to obtain neomycin resistance genes. Correct removal of neomycin resistance gene was confirmed by PCR of genomic DNA extracted from the tail of the mouse. In addition, the DNA obtained by PCR was sequenced to ensure that the neomycin gene was correctly removed. The primers used were as follows.

Exon2: 5'-GTCCAAGTGCAGAGATGAAGACGG-3 '(SEQ ID NO: 6),

AT2RV: 5'-CTCCCACTTTCCGATCTTAAGGGG-3 '(SEQ ID NO: 7).

< Example  8>

Mis18 α exons 1 and 2 removed Mis18 α gene Phlox  Production of mice (f / +)

Exons 1 and 2 of the Mis18α gene were removed from the Mix18α gene phlox mouse (f / +) to eliminate the Mis18α gene function in mice. Mis18α genes from which neomycin resistance genes have been removed Flux mouse (f / +) males and female mice with Cre recombinant enzymes regulated by protamine promoter (protamine-Cre, Jackson laboratory) Got. In this case, Cre recombinase is expressed at the stage of male sperm maturation, so that exons 1 and 2 of the Mis18α gene can be permanently removed from all later tissues and cells.

Among the pups, males having the Mis18α gene phloxed (f / +) genotype and expressing Cre recombinase at the same time were selected and crossed with females of C57BL / 6 species. Some of the offspring obtained at this time show exons 1 and 2 removed from one gene of Mis18α (Δ / +). Exon 1 and 2 were correctly removed from the Mis18α gene by PCR using genomic DNA extracted from the tail of the mouse.

In addition, DNA obtained by PCR was sequenced to ensure that exons 1 and 2 were correctly removed. The primers used were Exon2 (SEQ ID NO: 6) and AT2RV (SEQ ID NO: 7) as described above. When exons 1 and 2 were removed, a PCR product of about 200 bp was made. Finally it marks the exon 1 and 2. The genotypes of the mice were removed from one side of the gene in the genome to Mis18α Mis18 α ^{+ / Δ.}

< Example  9>

Mis18 α gene defect early Embryonic formation  Impact analysis at each stage

Mice that had exons 1 and 2 removed from one of the Mis18α genes in their genomes did not differ in appearance from normal mice and had no problems with growth and reproductive function. However, if both Mis18α genes are missing ( Mis18 α ^{Δ / Δ} Genotype) did not appear to be born normally. Mis18 α ^{+ / Δ} Mis18 α ^{Δ / Δ} out of 79 pups analyzed when genotyped mice females and males were bred. No genotypes could be found (Figures 8 and 9). This suggests that if both Mis18α genes are missing, mice die before birth.

To determine when embryos die at one stage of development, mice with one Mis18α gene cross are crossed with each other, and embryos are isolated from pregnant females at each developmental stage to determine when embryos with both Mis18α genes die. saw. Mis18 α ^{Δ / Δ on} days 12.5 and 9.5 of pregnancy No genotypes have been found alive. However, in embryos at 3.5 days of gestation, 20 of the 67 were Mis18 α ^{Δ / Δ} Genotypes were identified (FIGS. 8 and 10).

Thus, if both Mis18α genes were deleted, the embryos were found to survive until at least 3.5 days of pregnancy. In order to more precisely analyze the embryo mortality, mice with one side of the Mis18α gene were crossed with each other, and embryos were extracted at 3.5 days of pregnancy, and then artificially cultured for growth.

Embryos with normal Mis18α genes ( Mis18 α ^{+ / +} ) or single-sided ( Mis18 α ^{+ / Δ} ) embryos are peeled off and cultured to inner plates and trophoblasts within 48 hours of culture. It settled and began to grow. After about 72 hours, it was clearly observed that the endocytic cells divide to form agglomerates and grow normally up to about 92 hours (FIG. 11). However, embryos that lack both Mis18α genes have no problem at the stage where the embryos are peeled and the inner cells and trophoblasts settle in culture plates and begin to grow. It was confirmed that no lumps were formed (FIG. 11). Eventually the cells degenerated and did not remain in the culture dish at 92 hours.

Therefore, the present inventors concluded that if both Mis18α genes are deleted, the embryos will survive until 3.5 days of gestation and will die in the near term.

< Example  10>

Mis18 Analysis of the Effects of α Gene Defects on Chromosome Cleavage and Apoptosis

The inventors have Mis18 order to examine in more detail the cause of the Mis18α gene-deficient mice die in embryonic state α ^{+ / Δ} After mating female and male genotypes, embryos were extracted at 3.5 days of gestation and artificially cultured for 24 hours, and mitosis was analyzed by immunostaining.

Cells in mitosis were identified by immunostaining of histone H3 serine residues (H3S10 phosphorylation, a phenomenon specifically found only in mitotic DNA), and DAPI (4 '). , 6'-diamidino-2 -phenylindole) was used to stain the chromosomes, and the spindle was stained by immunostaining for alpha-tubulin (α-tubulin).

In the case of normal mouse embryos, spindles composed of chromosomes and microtubules were normally arranged and separated in the mitosis process of the early, middle, late, and late stages. Compared to that seen in normal mouse embryos (FIG. 12A), in the case of Mis18α-deficient mouse embryos, chromosomes and spindles were abnormally aligned (FIG. 12B). In embryonic cells with a normal Mis18α gene, the chromosomes were well aligned on the equator plane of the cells, and the shape of the spindle was connected to the chromosomes without errors (FIG. 13A). However, in the case of Mis18α-deficient mouse embryos, chromosomes are not properly arranged in the dividing cells and satellite chromosomes are cut off (FIG. 13B).

In addition, the spindle was not attached to the chromosome was found a lot, there was a problem that the chromosome centered around the mitosis, the chromosome was cut during the end of chromosome cleavage. The present inventors have found that Mis18α plays an important role in chromosome division during embryonic cell division, and in particular, it can be confirmed that Mis18α is a very important factor in the development of embryonic cells.

In addition, the present inventors have Mis18 α ^{+ / Δ} to determine whether the Mis18α play an important role in the process of which is located centromeric after the analog, CENP-A histone H3 newly synthesized After mating mice and males having genotype, embryos were extracted at 3.5 days of gestation and artificially cultured for 24 hours, followed by immunostaining for CENP-A protein, and chromosome staining with DAPI.

Since the CENP-A protein is located only in the isotope, it can be seen that when it is stained with an antibody, it is distributed as a dot in the cell nucleus. Although staining was observed, nuclei were stained with DAPI in the cells of Mis18α-deficient mouse embryos, but CENP-A did not show a spot staining in the nucleus (FIG. 14).

CENP-A is a histone analog that plays an important role in the formation of a centromere to which spindles are attached during chromosome cleavage. Cell death of the Mis18α-deficient mouse embryos was examined by TUNEL (Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling).

As a result, apoptosis was very low in normal mouse embryonic cells (1.8 ± 0.3 per embryo), whereas apoptosis in cells of Mis18α-deficient mouse embryos was statistically significantly higher (7.8 ± 1.5 per embryo) than in normal mouse embryonic cells. embryo) (FIG. 15). In conclusion, the deletion of the Mis18α gene prevented CENP-A from being located at the centromere, which leads to abnormal chromosomal division in cell division and eventually cell death. The present inventors were able to find out that embryos lacking the Mis18α gene can no longer develop into individuals due to apoptosis.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Attach an electronic file to a sequence list

Claims (14)

LoxP (locus of X-over P1) region, the gene segment from the second closest Eco RI cleavage site to the second exon in the 5 'direction of the mouse Mis8α genomic gene exon, the neomycin resistance gene and the Eco RI Mouse Mis18α gene targeting vector comprising a DNA sequence consisting of cleavage sites. The method of claim 1,
The neomycin resistance gene is a mouse Mis18α gene targeting vector, characterized in that in the order of the FRT (Flippase Recognition Target) site, LoxP site, neomycin resistance gene, FRT site and LoxP site. Embryo stem cell of the mouse of which the homologous recombination of the mouse Mis18α gene targeting vector of Claim 1 was introduce | transduced. A chimera mouse prepared by injecting embryonic stem cells of claim 3 into a blastocyst of a normal mouse and implanting the embryonic stem cells into a surrogate mother. Claim 4 of the mice Mis18α one gene phlox de (floxed) having a Mis18 α ^{f / +} genotype produced by mating the chimeric mice and normal mice. Claim 5 Mis18 α ^{f / +} genotype mice, with Cre Mis18α one gene deficient mice with the recombinant enzyme Mis18 α ^{+ / Δ} genotype produced by crossing mice expressing. Claim 6 Mis18α gene-deficient mouse embryo having Mis18 ^Δ α ^{/ Δ} genotype that is produced by mating the gene-deficient mice Mis18α one. In the method for producing a Mis18α ^{+ / Δ} transgenic mouse in which one Mis18α gene on the genome has been removed,
Preparing a mouse Mis18α gene targeting vector according to claim 1;
Introducing the targeting vector into embryonic stem cells to obtain an embryonic stem cell clone in which a part of the Mis18α gene has been removed by homologous recombination;
Injecting the Mis18α gene deficient embryonic stem cells into the inner cell mass of the blastocyte of a normal mouse and implanting the same into a surrogate mother mouse to obtain a chimera mouse; And
A method for producing a Mis18α gene deficient mouse, comprising the step of obtaining a Mis18α single gene deficient mouse having a Mis18α ^{+ / Δ} genotype through mating from the chimeric mouse. delete The method of claim 8,
The step of obtaining a Mis18α single gene deficient mouse having a Mis18α ^{+ / Δ} genotype through mating from the chimeric mice,
Mating the chimeric mice with mice expressing Flp recombinant enzyme (Flippase);
Selecting mice expressing the Flp recombinase from among the progeny obtained from the cross and at the same time showing the Mix18α one-side gene floxed (f / +) genotype;
Mating the selected mice with normal mice to obtain a mouse from which a neomycin resistance gene has been removed;
Mating the mouse from which the neomycin resistance gene has been removed with a mouse that expresses Cre recombinase;
Selecting mice expressing Cre recombinase at the same time of Mis18α gene phloxed (f / +) genotype among progeny obtained through the crossing; And
And mating the selected mouse with a normal mouse to obtain a mouse from which exons 1 and 2 are removed from one gene of Mis18α. delete 8. The method of claim 7,
The embryo is a Mis18α ^{Δ / Δ} transgenic mouse embryo, characterized in that the cell does not develop due to chromosomal division abnormalities and dies . Treating a Mis18α ^{Δ / Δ} embryo of claim 12 with a candidate for treating or preventing cancer or autoimmune disease; And
A method for screening a cancer or autoimmune disease therapeutic agent and a prophylactic agent comprising the step of comparing the cell division process of the untreated Mis18α ^{Δ / Δ} embryo treated with the therapeutic material and the material that restored the cell division ability.
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