JPWO2008084566A1 - Model mouse, manufacturing method thereof and use thereof - Google Patents

Abstract

A model mouse is provided for elucidating the function of Gsdmd and the Gsdmd gene encoding it in intestinal epithelial cells. A mouse having a homozygote obtained by knocking out a Gsdmd gene encoding Gsdmd on the chromosome and not expressing Gsdmd is defined as a model mouse. First, a targeting vector having a fragment containing exon 2 of the Gsdmd gene in mouse genomic DNA is introduced into a mouse embryonic stem cell, and a chimeric embryonic cell is produced from an embryonic stem cell in which the Gsdmd gene is knocked out by homologous recombination. This is transplanted into a temporary parent mouse, a chimeric mouse in which the born Gsdmd gene is knocked out is bred, a heterozygous mouse is selected, and the heterozygous mouse is bred with each other so that the Gsdmd gene is knocked out. Homozygous model mice are obtained.

Description

  The present invention relates to a model mouse and a manufacturing method thereof. More specifically, the present invention relates to a model mouse that can be used for analysis of Gasdermin (Gsdm) D and Gsdmd gene encoding the same in intestinal epithelial cells and a method for producing the same.

  Mutation Rim3 (Recombination-induced mutation 3) showing traits of epidermal hyperplasia, keratin growth and abnormal hair growth is known in mouse skin, and the present inventors have confirmed that the causative gene is Gsdma3 gene. It has been elucidated (see Patent Document 1). And by our further research, a total of 8 mouse genes including Gsdma3 gene, namely Gsdma1, Gsdma2, Gsdma3, Gsdmc1, Gsdmc2, Gsdmc3, Gsdmc4 and Gsdmd form an interrelated Gsdm family. It became clear that All the genes belonging to the Gsdm family have been confirmed to be specifically expressed in epithelial cells of various tissues including the skin and the digestive tract. From the Rim3 phenotype and the function of the Gsdma3 gene, it is speculated that each gene belonging to this family is involved in, for example, epithelial cell proliferation, differentiation, and epithelial morphogenesis.

However, the function of the Gsdmd gene expressed in intestinal epithelial cells such as the small intestine and the large intestine has not been elucidated at all. Corresponding to the mouse Gsdm family, the existence of the GSDM family is also known in human chromosomes. Therefore, elucidating the function of the mouse Gsdmd gene is extremely useful information for elucidating the function of the human GSDM gene.
WO 2004/0776666 (PCT / JP03 / 02345) Katoh, M .; and Katoh, M .; Identification and characterization of DFNA5L, mouse Dfna5l and rat Dfna5l genes in silico. Int J oncol. 25, 765-770, 2004.

  Accordingly, an object of the present invention is to provide a model mouse for elucidating the functions of Gsdmd and the Gsdmd gene encoding it in intestinal epithelial cells.

  In order to achieve the above object, the model mouse of the present invention is a model mouse in which a gene is knocked out, and the mouse is a homozygote in which a Gsdmd gene is knocked out on a chromosome.

The production method of the present invention is a method of producing a model mouse in which a gene is knocked out, wherein the model mouse is the model mouse of the present invention and includes the following steps (A) to (H): .
(A) Step of preparing a targeting vector having a fragment containing the genomic region of the Gsdmd gene in mouse genomic DNA (B) Introducing the targeting vector into mouse embryonic stem cells, and homologous recombination between mouse genomic DNA and the targeting vector (C) A step of selecting an embryonic stem cell in which the Gsdmd gene is knocked out by the homologous recombination (D) A step of producing a chimeric embryo cell from the embryonic stem cell obtained in the step (C) (E) The chimera Step of transplanting embryonic cells into temporary parent mice (F) Step of selecting chimeric mice born from the temporary parent mice and knocked out of the Gsdmd gene (G) Step of mating the chimeric mice and selecting heterozygous mice (H) Crossing the heterozygous mice to produce Gsdmd Step gene to select a knocked-out homozygous mice

The drug screening method of the present invention is a method for screening a drug that restores the cell proliferative function of a damaged cell, and includes the following steps (a) to (c).
(A) a step of preparing a model mouse of the present invention damaged in the intestine (b) a step of administering a candidate substance to the model mouse (c) improving the cell proliferation function in the damaged intestine of the model mouse Of selecting candidate substances as target drugs

  The model mouse of the present invention is a homozygote in which an endogenous Gsdmd gene on the chromosome is knocked out. Therefore, by using this model mouse, for example, functional analysis of Gsdmd and Gsdmd genes in intestinal epithelial cells can be performed. In addition, it has been clarified by the present inventors that the model mouse of the present invention exhibits a trait in which the proliferative function of intestinal cells is reduced, for example, when the intestine is damaged. Therefore, by using the model mouse of the present invention in which the intestine is further damaged, for example, it is possible to screen for a drug that restores the cell growth function. In addition, since the cell proliferation function in the tissue after injury is a function responsible for homeostasis as described later, the model mouse of the present invention is an extremely useful tool in elucidating homeostasis of intestinal tissue, for example.

FIG. 1 is a diagram showing an outline of homologous recombination in Example 1 of the present invention. The genomic structure of the mouse Gsdmd gene, the structure of a targeting vector, the structure of a recombinant allele, and the Gsdmd null allele (Cre An outline of (deleted allele) is shown. FIG. 2 shows the results in Example 1 of the present invention, (B) shows the results of genotyping of the recombinant ES cells, and (C) shows the genotyping of Gsdmd null allele. It is a figure which shows a result, (D) is a figure which shows the expression level of Gsdmd gene and (beta) -actin gene. FIG. 3 is photographs of intestinal tracts of Gsdmd null mutant mice in Example 2 of the present invention, and (A) and (C) are photographs of small intestine (A) and large intestine (C) of Gsdmd null mutant mice. (B) and (D) are stained photographs of the small intestine (B) and large intestine (D) of wild-type mice. FIG. 4 is a stained photograph of the mouse intestinal tract in Example 2 of the present invention, and the left column (A, C, E, G) is a stained photograph of the small intestinal epithelium of a Gsdmd null mutant mouse, and the right column ( B, D, F, H) are stained photographs of the small intestinal epithelium of wild type mice. FIG. 5 is a stained photograph of the mouse intestine in Example 2 of the present invention, the left column (A, C) is a stained photograph of the colon epithelium of a Gsdmd null mutant mouse, and the right column (B, D). These are stained photographs of the colon epithelium of wild-type mice. FIG. 6 shows the results of analysis of cell proliferation and apoptosis in the small intestine of Gsdmd null mice in Example 3 of the present invention. (A) and (B) are the results of immunohistochemical staining showing cell proliferation, (C) and (D) is the result of immunohistochemical staining showing apoptosis. FIG. 7 shows analysis results of cell proliferation and apoptosis of Gsdmd null mouse large intestine in Example 3 of the present invention. (A) and (B) are the results of immunohistochemical staining showing cell proliferation. (C) And (D) are the results of immunohistochemical staining showing apoptosis. FIG. 8 is a graph showing the relative expression levels of mRNA of Gsdmc2 gene, Gsdmc3 gene, and Gsdmc4 gene in Example 4 of the present invention. FIG. 9 is a graph showing the relationship between the survival time and mortality of Gsdmd null mice in Example 5 of the present invention. FIG. 10 shows the results of apoptosis in mouse small intestine after γ-irradiation in Example 6 of the present invention, (A) is a graph of apoptotic cells / crypts, and (B) is Gsdmd null mouse small intestine. The photograph (C) is a photograph of the wild-type mouse small intestine. FIG. 11 shows the results of apoptosis in mouse colon after γ-irradiation in Example 6 of the present invention, (A) is a graph of apoptotic cells / crypts, and (B) is Gsdmd null mouse colon. The photograph (C) is a photograph of the wild-type mouse colon. FIG. 12 shows the results of cell proliferation in the mouse small intestine after γ-irradiation in Example 6 of the present invention, where (A) is a graph of BrdU positive cells / crypts, and (B) is a Gsdmd null mouse. A photograph of the small intestine, (C) is a photograph of the wild-type mouse small intestine. FIG. 13 shows the results of cell proliferation of mouse colon after γ-irradiation in Example 6 of the present invention, (A) is a graph of BrdU positive cells / crypts, and (B) is a Gsdmd null mouse. A photograph of the large intestine, (C) is a photograph of the wild-type mouse large intestine. FIG. 14 is a bar diagram showing the frequency distribution of BrdU positive cells in the mouse small intestine after γ-irradiation in Example 6 of the present invention. FIG. 15 is a bar diagram showing the frequency distribution of the number of BrdU positive cells in the mouse colon after γ-irradiation in Example 6 of the present invention. FIG. 16 shows the chromosomal location of the mouse Gsdmd gene. FIG. 17 is a diagram showing the genomic structure of the mouse Gsdm family on chromosome 15.

  As described above, the model mouse of the present invention is a model mouse in which a gene is knocked out, and is a homozygote in which a Gsdmd gene is knocked out on a chromosome. In the present invention, “Gsdmd gene knockout” is to eliminate the function of the endogenous Gsdmd gene on the chromosome. For example, the Gsdmd gene is inactivated by gene mutation and the function of expressing Gsdmd is lost. Means that.

  The method of gene mutation is not limited, and examples thereof include deletion, substitution, and addition of the Gsdmd gene. Further, the function of the Gsdmd gene (for example, the function of expressing Gsdm) may be lost by gene mutation, and for example, the mutation of the entire Gsdmd gene may be used, or a partial mutation may be used. That is, for example, the entire Gsdmd gene on the chromosome may be deleted, replaced or added, or partially deleted, replaced or added. As a specific example, since exon 2 of the Gsdmd gene contains a translation initiation site (ATG), for example, exon 2 is preferably deleted, substituted or added, and a region containing exon 1 and exon 2 It is also preferred that is deleted, substituted or added. In addition to this, for example, the transcriptional regulatory region of the Gsdmd gene (5s upstream genomic region of Gsdmd exon 1) may be deleted.

  Since the model mouse of the present invention is a homozygote in which the Gsdmd gene is knocked out, the gene product (mRNA, protein) of the Gsdmd gene exhibits a trait that it is not expressed. In addition, as a result of repeated studies on the obtained model mice of the present invention, the present inventors have revealed the following traits. That is, the model mouse of the present invention exhibits a trait in which the intestinal cell proliferative function is reduced when the intestine is damaged. In general, when a tissue is damaged, the living body attempts to regenerate the tissue and maintain homeostasis by apoptosis of the damaged cell and improvement of the proliferation rate of the cell (stem cell). For this reason, it can be said that the cell proliferation function in the injured tissue is responsible for homeostasis. As described above, the mouse of the present invention has a decreased intestinal cell proliferative function. From this, the Gsdmd gene is presumed to be responsible for, for example, homeostasis of intestinal epithelial cells in the mature stage, not cell differentiation or tissue morphogenesis.

  Thus, when the intestinal tissue is damaged, the model mouse of the present invention exhibits a trait in which the cell proliferation function is reduced. Therefore, the model mouse of the present invention in which the intestinal tissue is damaged is useful as, for example, a model mouse for screening a drug that restores the cell proliferation function, as described later.

  As described above, the model mouse of the present invention has a homozygote in which the Gsdmd gene is knocked out, and Gsdmd may be unexpressed. For this reason, although the manufacturing method is not restrict | limited, For example, it can manufacture by the model mouse manufacturing method of this invention. Below, an example is given and demonstrated about the manufacturing method of the model mouse | mouth of this invention. The present invention is not limited to this.

  First, a targeting vector having a fragment containing exon 2 of the Gsdmd gene in mouse genomic DNA, preferably a fragment containing exon 2 and exon 1 (including introns between exons) is prepared (step A). The method for preparing this fragment is not limited, and a conventionally known method can be adopted. Specific examples include a method of cloning from a mouse gene library (genomic DNA) and a method of amplifying a target fragment by PCR or the like. The sequence of the Gsdmd gene in mice is described in, for example, NCBI Accession No. Registered in Ak007710. An outline of the position of the mouse Gsdmd gene on chromosome 15 is shown in FIG. 16, and an outline of the genome structure is shown in FIG. In both figures, the type of Gsdm gene is described in capital letters GsdmC1 to GsdmC4 and GsdmD, but these are Gsdmc1 to Gsdmc4 and Gsdmd, respectively, and the same applies to the other figures.

  The targeting vector preferably has a cassette in which both ends of the fragment are sandwiched between recognition sequences for Cre recombinase. The recognition sequence of Cre recombinase is not particularly limited, and examples thereof include conventionally known loxP sequences. If loxP is used, gene targeting can be easily performed by a so-called Cre-loxP recombination system. Moreover, it is preferable that the cassette further includes a selection marker. Examples of the selection marker include drug resistance markers and fluorescent protein markers. Examples of the drug resistance marker include a neomycin resistance marker, a zeocin resistance marker, a puromycin resistance marker, a hygromycin resistance marker, a histidinol resistance marker, and the like. By including such a selection marker, for example, selection of embryonic stem cells (ES cells) described later is facilitated.

  Next, the targeting vector is introduced into mouse ES cells, and homologous recombination between mouse genomic DNA and the targeting vector is performed (step B). The introduction of the targeting vector into ES cells can be appropriately determined depending on the type of vector, for example. A specific example is a method in which the targeting vector is linearized and introduced into ES cells by electroporation or the like.

  Then, ES cells in which a Cre recombinase recognition sequence (for example, loxP sequence) is inserted into the Gsdmd gene by the homologous recombination are selected (step C). As a specific example, when a targeting vector in which both ends of the aforementioned exon 2 and exon 1-containing fragment are sandwiched between loxP sequences is used, for example, upstream of exon 1 of Gsdmd gene and intron 2 (between exon 2 and exon 3, And so on), and ES cells into which the loxP sequence has been inserted are selected. The method of selection is not limited at all. As described above, when the targeting vector has the selection marker, for example, ES cell culture may be performed and an expression strain of the selection marker may be selected. This selectable marker expression strain is a recombinant ES cell line having an allele into which the cassette has been introduced by homologous recombination. When the selection marker is a drug resistance marker, a drug resistant strain may be selected.

  The obtained recombinant ES cell is injected into, for example, a mouse embryo cell (blastocyst) to produce a chimeric embryo cell (D step), and this chimeric embryo cell is transplanted into the uterus of a temporary parent mouse (E step) ). Then, a chimeric mouse having the loxP sequence inserted into the Gsdmd gene is selected from the mouse born from the foster parent mouse (step F). As a specific example, when the above-mentioned ES cell in which a loxP sequence is inserted upstream of exon 1 and intron 2 of the Gsdmd gene is used, a chimera in which a loxP sequence is inserted upstream of exon 1 and intron 2 of the Gsdmd gene. Sort mice. This selection can be performed, for example, by adjusting the combination of mouse strains to be used and discriminating from the appearance based on the hair color of the born mouse.

  The chimeric mice are crossed to select heterozygous mice (Step G). This selection can be performed, for example, by selecting an appropriate PCR primer and performing PCR using the born mouse genomic DNA as a template. When the Gsdmd gene fragment is sandwiched between Cre recombinase recognition sequences (loxP sequences) in the targeting vector, for example, a transgenic mouse expressing Cre and the chimeric mouse may be crossed. Then, the obtained heterozygous mice are mated to select a homozygous mouse in which the Gsdmd gene is knocked out. In this way, the model mouse of the present invention can be manufactured.

  Further, as described above, the model mouse of the present invention may further be a mouse in which the intestine is damaged. For this reason, the homozygous mouse obtained as described above may be further subjected to treatment such as gamma irradiation, drug exposure, and bacterial infection. The model mouse of the present invention in which the intestine is damaged by such treatment shows a trait that the cell proliferation function is reduced in the intestinal tissue. Such a model mouse is useful for screening described later, for example. Examples of the drug include, but are not limited to, ethanol, acetic acid, hydrochloric acid, sodium dextran sulfate, indomethacin, trinitrobenzene sulfonic acid, peptidoglycan-polysaccharide, immune complex / formalin, cyclosporine and the like. Examples of the bacterium include H. pylori.

Next, as described above, the screening method of the present invention is a screening method for a drug that restores the cell proliferation function of damaged cells, and includes the following steps (a) to (c): To do.
(A) Step of preparing a model mouse of the present invention damaged in the intestine (b) Step of administering a candidate substance to the model mouse (c) A candidate substance having improved cell proliferation function in the intestine of the model mouse , The process of selecting as the desired drug

  According to this method, it is possible to screen for a drug that restores the decreased cell proliferation function. Since the drug screened in this way can restore or enhance the cell proliferative function responsible for homeostasis, it is very useful, for example, in the medical field.

  Next, examples of the present invention will be described together with comparative examples. However, the present invention is not limited by the following examples.

[Example 1]
Production of Gsdmd Gene Null Mutant Mice For mouse ES cells, exon 1 and exon 2 of the Gsdmd gene were deleted by homologous recombination to produce null mutant mice lacking the Gsdmd gene product. Exon 2 of the Gsdmd gene contains ATG as the translation start site.

  C57BL / 6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA), MCH (Multi Cross Hybrid (ICR)) strain mice and B6C3F1 (F1 of C57BL / 6J and C3H strains) strains were CLEA JAPAN, IN . (Tokyo, Japan).

(1) Preparation of targeting vector First, as a basic targeting vector, ploxFNFDDT containing 5'loxP-flrted Pgk (phosphoglycerate kinase) -neo-3'loxP and Pgk-DTA (diphtheria toxin A subunit) was prepared. The ploxFNNFDT is a multicloning site of Stratagene pBluescript II SK (+), 5′-loxP sequence / FRT sequence / Pgk (phosphoglycerate kinase) promoter / neomycin resistance gene / FRT sequence / loxP sequence-3 ′, This is a vector into which Pgk-DTA (diphtheria toxin A subunit) has been introduced. On the other hand, as a fragment of the Gsdmd gene of the mouse genome, a 5 ′ flanking fragment (5.4 kb), a fragment containing exons 1 and 2 (3.5 kb), a fragment containing exons 3 and 4 (1.4 kb) are designated as C57BL. / 6J mouse BAC clone (BAC RPCI24 340J12) was prepared by a conventional method. Hereinafter, a fragment containing exons 1 and 2 is referred to as “exon 1-2 fragment”, and a fragment containing exons 3 and 4 is referred to as “exon 3-4 fragment”. In the ploxFNFDT, the 5 ′ flanking fragment is upstream of the 5′-loxP, the exon 1-2 fragment is inserted between the 5′-loxP and the fluttered Pgk-neo site, and the 3′-loxP downstream. The exon 3-4 fragment was bound to each other. This vector was used as a targeting vector. FIG. 1A shows an outline of the genomic structure of the mouse Gsdmd gene and the structure of the targeting vector. In the figure, Ex is an exon, DT is DTA (coding sequence of diphtheria toxin A subunit), Neo is a neomycin resistance gene, loxP represented by a large arrow is a target sequence of the recombinase Cre, and is represented by a vertical ellipse. The FRTs indicated indicate the target sequence (flrtted) of the recombinant enzyme Flp, respectively. Moreover, the small arrow in the same figure shows the various primers mentioned later. As shown in FIG. 1A, the prepared targeting vector has a region where exon 1-2 fragment and neo cassette are sandwiched by loxP.

(2) Gene targeting TT2 cell line (Yagi et al., Anal. Biochem. 214, 70-76, 1993) established from F1 mice obtained by crossing C57BL / 6J mice with CBA / J mice, Used as ES cells. The linearized targeting vector was introduced into the ES cells by electroporation, and neomycin (G418) resistant strains were selected (a total of 218 strains). Furthermore, a recombinant ES cell line having a recombinant allele homologously recombined with the targeting vector was screened from the G418 resistant strain by the following method (11 strains). An outline of this recombinant allele is shown in FIG. 1A.

  The G418 resistant strain was screened by PCR using the following two primer sets (ESPCR1, ESPCR2). PCR conditions were set based on conventional methods. An outline of the annealing site for the genomic DNA of each primer set is shown together with the recombinant allele of FIG. 1A. This screening was performed by first selecting a strain whose amplification was confirmed by the ESPCR1 primer set, and further selecting a strain whose amplification was confirmed by the ESPCR2 primer set. FIG. 2B shows the result of genotyping the recombinant ES cells using the primer sets ESPCR1 and ESPCR2. In FIG. 2B, the upper panel shows the band amplified by the ESPCR1 primer set, and the lower panel shows the band amplified by the ESPCR2 primer set. A genomic DNA having a wild type allele (homozygote: + / +) does not have a 1688 bp fragment (exon 1-2 fragment and neo cassette sandwiched by loxP). For this reason, as shown in the upper panel left lane of FIG. 2B, the amplification band by the ESPCR1 primer set was not confirmed, and as shown in the lower panel left lane, the amplification band by the ESPCR2 probe set (1080 bp not including 5′loxP) Was only confirmed. In contrast, genomic DNA (heterozygote: + / flox) having a recombinant allele was introduced with a 1688 bp fragment (exon 1-2 fragment and neo cassette sandwiched by loxP). As shown in the right lane of the upper panel of 2B, an amplification band by the ES PCR1 primer set was confirmed. Furthermore, as shown in the lower lane on the left panel, an amplified band (1100 bp fragment containing 5'loxP) by the ES PCR2 probe set was also confirmed.

ESPCR1 primer set P1F
5′-GAGGATTGGGAAGACAATAG-3 ′ (SEQ ID NO: 1)
P1R
5′-AGGTCCACCACTACTACTATCC-3 ′ (SEQ ID NO: 2)
ESPCR2 primer set P2F
5′-GCATGCCGTAGTTGTGCACTT-3 ′ (SEQ ID NO: 3)
P2R
5′-GGCTATAGAGATGGGCCCTGCAGTT-3 ′ (SEQ ID NO: 4)
* F is forward primer, R is reverse primer

  The obtained G418-resistant recombinant ES cell line was injected into an 8-cell embryo and transplanted into the uterus of a temporary parent mouse (B6C3F1). A male chimera born from the foster parent mouse was crossed with an MCH female mouse, and the transfer of the recombinant allele was confirmed depending on whether or not the hair color of the born mouse was black. Among mice born by mating, heterozygous mutant mice having the recombinant allele introduced with loxP are B6. A heterozygous mutant mouse having a Gsdmd-null allele was obtained by mating with a Tg (CAG-Cre) transgenic mouse. The heterozygous mutant mice and B6. A mouse born by mating with a Tg (CAG-Cre) transgenic mouse expresses Cre, thereby causing recombination between loxP sequences. An outline of the Gsdmd null allele (Cre-deleted allele) in which this recombination has occurred is shown together with FIG. 1A.

Then, heterozygous mutant mice (Gsdmd ^+/− ) having Gsdmd null alleles were mated to obtain homozygous mutant mice (Gsdmd ^{− / −} ). Hereinafter, the homozygous mutant mouse is also referred to as a Gsdmd null mutant mouse.

  For the mice obtained by mating, genotyping of the Gsdmd null allele was performed by PCR using mouse tail-derived genomic DNA and the following two types of primer sets (gtPCR1, gtPCR2). The outline of the annealing site for the genomic DNA of each primer set is shown together with Gsdmd and Cre-deleted allele in FIG. 1A. The result of this genotyping is shown in FIG. 2C. In FIG. 2C, the upper panel shows the band amplified by the gtPCR1 primer set, and the lower panel shows the band amplified by the gtPCR2 primer set. In the genomic DNA (wild type + / +) having the wild type allele, as shown in the upper panel left lane of FIG. 2C, only an amplification band of 612 bp by the gtPCR1 primer set was confirmed. On the other hand, in the genomic DNA (homozygote-/-) having the Gsdmd null allele, the amplification band by the gtPCR1 primer set is not confirmed, and the amplification band by the gtPCR2 probe set (943 bp) is confirmed as shown in the right lane of the lower panel It was.

gtPCR1 primer set gt1F
5′-TTCCAAATCCACAGCCTAAGAG-3 ′ (SEQ ID NO: 5)
gt1R
5′-AGCTCAATAAATAAACAAGAC-3 ′ (SEQ ID NO: 6)
gtPCR2 primer set gt1F
5'- TTCCAATCCACAGCCTAAGAG-3 '(SEQ ID NO: 7)
gt2R
5- AGCTCAATAAATAAACAAGAC-3 ′ (SEQ ID NO: 8)

Moreover, when the generation | occurrence | production of the homozygous mutant mouse | mouth (Gsdmd <^-/-> ) was confirmed, as shown in following Table 1, it turned out that it generate | occur | produces with the isolation | separation ratio of the usual Mendelian.

The resulting Gsdmd null mutant mice were indistinguishable from wild type and heterozygous mutant mice. Therefore, for each mature mouse (wild-type mouse Gsdmd ^{− / −} , heterozygous mutant mouse Gsdmd ^+/− , Gsdmd null mutant mouse Gsdmd ^{− / −} ), the expression level of Gsdmd gene in the small intestine was determined using the following primer set: Analyzed by RT-PCR. As a control, the expression level of β-actin was similarly analyzed. These results are shown in FIG. 2D. As shown in FIG. 2D, the expression of mRNA of Gsdmd was confirmed in the wild type mouse and the heterozygous mutant mouse, but the expression of mRNA was not confirmed in the Gsdmd null mutant mouse. From this result, it was confirmed that the obtained Gsdmd null mutant mouse was deficient in its gene product due to partial deletion of the Gsdmd gene. As described above, homozygous Gsdmd mutant mice lacking the Gsdmd gene product could be obtained.

Primer set for Gsdmd Gsdmd-F
5′-CATGGCCCTCAATGTGCTTGC-3 ′ (SEQ ID NO: 9)
Gsdmd-R
5′-GGTGGGCAGGGACACAGAAC-3 ′ (SEQ ID NO: 10)
Primer set for β-actin β-actin-F
5′-GCCATCCTGCGTCTGGAACT-3 ′ (SEQ ID NO: 11)
β-actin-R
5′-AGGAAGAGGATGCGGCAGTG-3 ′ (SEQ ID NO: 12)

[Example 2]
(Confirmation of Gsdmd null mouse expression system)
The Gsdmd gene is mainly expressed in the small and large intestines of mature mice. Therefore, histological analysis by staining was performed on the small and large intestines of Gsdmd null mutant mice (Gsdmd ^{− / −} ) to confirm the expression system.

First, from the two month old mouse, the entire intestinal tract was taken out and washed with a gentle flow of phosphate buffered saline (PBS) to remove the contents. The small intestine and large intestine after washing were each wound in a circular shape and fixed overnight at 4 ° C. in PBS containing 4% by volume PFA (paraformaldehyde). This was embedded in paraffin, and a 7 μm thick paraffin section was prepared. The sections were deparaffinized with xylene, rehydrated with ethanol, and then stained with HE (hematoxylin / eosin). A similar analysis was performed for wild-type mice (Gsdmd ^{+ / +} ). As wild-type mice, heterozygous mutant mice Gsdmd ^+/− were mated with each other, and gsdmd null mutant mice (Gsdmd ^{− / −} ) were used together with genetically normal littermate Gsdmd ^{+ / +} (hereinafter, referred to as “wild type mice”). The same).

  These results are shown in FIG. In FIG. 3, A and C are photographs of small intestine (A) and large intestine (C) of Gsdmd null mutant mice, and B and D are photographs of small intestine (B) and large intestine (D) of wild-type mice. (All are 200 times magnification). As can be seen from these figures, the Gsdmd null mutant mice showed no difference in histological characteristics in both the small intestine and the large intestine compared to the wild type mice. This result suggests that the Gsdmd gene is not involved in the development and morphogenesis of the small and large intestines, at least until the second month of life.

(Effects of Gsdmd gene on cell differentiation)
As described above, the inventors have confirmed that the Gsdm3 gene, which is one member of the Gsdm gene family, causes abnormalities in epithelial cell differentiation (Rim3 phenotype) by mutation. Therefore, cell differentiation was analyzed for the small intestine and large intestine of Gsdmd null mutant mice (Gsdmd ^{− / −} ), and the effect of the Gsdmd gene on cell differentiation was confirmed. Analysis of cell differentiation was performed by histological analysis by staining and immunohistochemical analysis.

  The intestinal epithelium is composed of four differentiated cells: goblet cells, panate cells, enterocytes, and enteroendocrine cells. Therefore, in this example, these four differentiated cells were confirmed by the following method. The following histological staining methods and immunohistological methods using antibodies specific for each cell were performed based on conventionally known methods unless otherwise indicated.

  Specifically, as described above, deparaffinized and hydrated sections (small intestine and large intestine) were stained with periodate Schiff (PAS), and immunohistochemistry using a primary antibody was performed. went. A PAS staining method was adopted for detection of goblet cells and panate cells. Intestinal cells and enteroendocrine cells employ an immunohistochemical method using a primary antibody. As the primary antibody, the intestinal cells include a rabbit anti-rat liver fatty acid binding protein (Fabp-L) antibody (dilution ratio 1: 100). ; Novus Biologicals) and rabbit anti-chromogranin A + B (Chga) antibody (dilution ratio 1: 200; manufactured by Progen) were used for enteroendocrine cells. As a solvent for diluting the primary antibody, a blocking solution (1.5% goat serum / PBS) was used (hereinafter the same).

The former PAS staining was performed based on a conventional method. The latter immunohistochemistry was performed as follows. First, antigen activation treatment was performed on the deparaffinized and hydrated sections prepared as described above. A commercially available antigen unmasking solution (dilution ratio 1: 100; manufactured by Vector Laboratories) was used for this antigen activation treatment. Specifically, the sections were incubated in the solution at 105 ° C. for 15 minutes in an autoclave. Then, the treated sections were incubated in 3% H ₂ O ₂ for 10 minutes, and further, the primary antibody was added and incubated at 4 ° C. overnight. Subsequently, this section was subjected to immunohistochemical staining by an ABC method using a staining kit (trade name: Vector Stain ABC kit, manufactured by Vector Laboratories). In addition, 1.5% goat serum / PBS was used for the blocking treatment of the antibody, and all operations were performed based on the instruction manual of the kit (Vector Stain ABC kit). The color reaction was carried out using a diaminobenzidine (DAB) / 50 mM Tris-HCl (pH 7.5) solution. In addition, the wild type mouse (Gsdmd ^{+ / +} ) was similarly analyzed.

  These results are shown in FIG. 4 and FIG. In FIG. 4, the left column (A, C, E, G) is a photograph of a small intestine epithelium of a Gsdmd null mutant mouse, and the right column (B, D, F, H) is a small intestine epithelium of a wild type mouse. It is a dyeing | staining photograph. In FIG. 4, the enlargement ratios of A to D, G, and H are 100 times, and the enlargement ratios of E and F are 200 times. In FIG. 4, detection signals for Fabp-L in A and B are enterocytes, PAS staining in C and D is goblet cells, PAS staining in E and F (signals indicated by arrows) is in panate cells, G and H Chga detection signals indicate enteroendocrine cells, respectively. In FIG. 5, left columns (A, C) are photographs of colon epithelium of Gsdmd null mutant mice, and right columns (B, D) are photographs of colon epithelium of wild type mice. The magnification in FIG. 5 is 100 times. In FIG. 5, PAS staining in A and B indicates Panate cells, and Chga detection signals in C and D indicate enteroendocrine cells, respectively. As shown in these figures, no significant difference was observed in the expression of these markers in the small intestine epithelium and large intestine epithelium of Gsdmd null mice and wild type mice. From these results, it was suggested that the function of the transcript of the Gsdmd gene is not involved in the final differentiation of the small and large intestines.

[Example 3]
(Effect of Gsdmd gene on cell proliferation and apoptosis)
It has been confirmed by the present inventors that the mutation of the Gsdma3 gene causes excessive cell proliferation in addition to the aforementioned abnormal differentiation of epithelial cells. Therefore, cell growth and apoptosis were analyzed for the small intestine and large intestine of Gsdmd null mutant mice (Gsdmd ^{− / −} ), and the influence of the Gsdmd gene was confirmed. Analysis of cell differentiation was performed by histological analysis by staining and immunohistochemical analysis.

(Analysis of cell proliferation)
The cell growth rate of Gsdmd null mice was confirmed by detecting bromodeoxyuridine (BrdU) incorporated into cellular DNA in the S phase. First, BrdU (50 mg / mouse kg) was injected into the peritoneum of a 2-month-old mouse, and after 2 hours from the injection, the small and large intestine sections were collected from the mouse in the same manner as in Example 2 above. Sections were prepared. In order to denature DNA, the section subjected to deparaffinization and hydration treatment in the same manner as in Example 2 was treated with 2N hydrochloric acid at 37 ° C. for 20 minutes, and antigen activation was performed with trypsin. This was performed at 3 ° C. for 3 minutes. Immunohistochemical staining was performed in the same manner as in Example 2 except that mouse anti-BrdU (dilution ratio 1: 1000; manufactured by Sigma) was used as the primary antibody. In addition, the wild type mouse (Gsdmd ^{+ / +} ) was similarly analyzed.

(Analysis of apoptosis)
Apoptosis of the cells was confirmed by detecting active caspase 3 which is a marker of apoptosis. Specifically, immunohistochemical staining (Nicholson et al, Nitroson et al.) Was carried out in the same manner as in Example 3 except that rabbit anti-cleavable caspase 3 (dilution ratio 1: 200; manufactured by Cell Signaling Technology) was used as the primary antibody. Nature 376, 37-43, 1995). In addition, the wild type mouse (Gsdmd ^{+ / +} ) was similarly analyzed.

  These results are shown together with FIG. 6 and FIG. FIG. 6 shows the results of analysis of cell proliferation and apoptosis in the small intestine. In FIG. 6, A and B are the results of immunohistochemical staining showing cell proliferation (magnification rate 100 times), and C and D are the results of immunohistochemical staining showing apoptosis (magnification rate 200 times). The left column shows the results of Gsdmd null mice and the right column shows the results of wild type mice. FIG. 7 shows analysis results of cell proliferation and apoptosis in the large intestine. In FIG. 7, A and B are the results of immunohistochemical staining showing cell proliferation (magnification 200 times), and C and D are the results of immunohistochemical staining showing apoptosis (magnification 200 times). The left column shows the results of Gsdmd null mice and the right column shows the results of wild type mice. As shown in FIGS. 6 and 7, no significant difference was observed between the Gsdmd null mouse and the wild-type mouse for the proliferating cells incorporating BrdU and the active caspase 3 positive cells showing apoptosis. From these results, it was suggested that the Gsdmd null mutant mice had normal cell proliferation and apoptosis in the small and large intestines, and that the Gsdmd gene and gene product did not affect cell proliferation and apoptosis.

[Example 4]
(Gsdmc cluster gene expression control)
The present inventors have confirmed that the same Gsdm gene family, Gsdmc cluster gene, is expressed in the crypt region of the intestine adjacent to the Gsdmd expression region. Therefore, the expression of the Gsdmc cluster gene in Gsdmd null mice was confirmed, and the influence of the Gsdmd gene on the expression of the Gsdmc cluster gene was confirmed.

The expression of the Gsdmc cluster gene was performed by a real-time PCR (RT-PCR) method using a TaqMan (registered trademark) probe. Total RNA is extracted from 2-month-old Gsdmd null mice using a reagent (trade name ISOGEN, manufactured by Nippon Gene Co., Ltd.) and further purified using the product name RQ1 DNase (produced by PROMEGA). It was prepared by. RT-PCR template cDNA was generated from 1 μg of the total RNA by PCR using the trade name Superscript (registered trademark) III and the trade name random hexamer primer (manufactured by Invitrogen). In the PCR, amplification under the following conditions was performed three times. In one PCR, treatment was performed at 50 ° C. for 2 minutes and at 95 ° C. for 10 minutes, and then 50 cycles were repeated with 95 ° C. for 15 seconds and 60 ° C. for 1 minute. The obtained cDNA was used as a template, and a primer-probe set specific for each gene of the Gsdmc cluster gene was used for quantification by RT-PCR based on conventionally known conditions. The primer-probe set is a commercially available product (Applied Biosystems, catalog No. 43194143E). For Gsdmc4, the product number is Mm01194497 g1. In addition, an 18S ribosomal RNA assay kit (Applied Biosystems probe / primer set, catalog No. 43194143E) was used for standardization of input cDNA variations. In addition, the relative level of gene expression was detected using the trade name ABI Prism 7700 Sequence Detection System (Applied Biosystems) and TaqMan Universal PCR Master Mix Reagent (Applied Biosystems), and Delta-Delta Ct (See Applied Biosystems User Bulletin # 2 ABI PRISM 7700 Sequence Detection System). For wild-type mice, heterozygous mutant mice Gsdmd ^+/− were mated with each other, and total RNA was prepared from the genotype normal litter Gsdmd ^{+ / +} obtained at the same time as the Gsdmd null mutant mouse (Gsdmd ^{− / −} ). The analysis was performed in the same manner except for the above.

  These results are shown in FIG. FIG. 8 is a graph showing the relative expression levels of mRNA of the Gsdmc2 gene, the Gsdmc3 gene, and the Gsdmc4 gene. As for the expression level, the expression level of wild-type mice was taken as 1, and the relative value was shown. As shown in the figure, the expression level of the Gsdmc cluster gene was significantly reduced in the Gsdmd null mouse compared to the wild type mouse. This is presumed that the expression of the Gsdmc cluster gene is down-regulated due to the deletion of the Gsdmd gene. From this result, it was found that the lack of specific phenotypes in the small and large intestines of Gsdmd null mice was not due to the influence of the Gsdmc cluster gene.

[Example 5]
(Gsdmd null mouse mortality)
The mortality rate of Gsdmd null mice from 1 month to 6 months after birth was confirmed. The confirmation of mortality was confirmed for 24 Gsdmd null mice (Gsdmd ^{− / −} ), 45 heterozygous mutant mice (Gsdmd ^+/− ) and 34 wild type mice (Gsdmd ^{+ / +} ).

  The result is shown in FIG. FIG. 9 is a graph showing the relationship between survival time (Month) and mortality, with homozygous mutant mice (Gsdmd null mice) marked with ◆, heterozygous mutant mice marked with ■, and wild-type mice marked with ▲. As shown in FIG. 9, the Gsdmd null mouse had a very high mortality rate compared to the heterozygous mutant mouse and the wild type mouse. Specifically, mortality at 12 months of age is 5.9% (2/34) and 11.1% (5/45) for wild-type and heterozygous, respectively, whereas homozygous The body showed 29.2% (7/24). The death rate at this age is also high. In addition, when the activity and appearance of the mouse were confirmed, no abnormality was particularly confirmed in the Gsdmd null mouse 24 hours before death.

  Thus, Gsdmd null mice lacking Gsdmd expression have a pronounced frequency of sudden death. From this, it is speculated that the function of Gsdmd is involved in intestinal epithelial homeostasis at maturity rather than the development and morphogenesis of the digestive tract.

[Example 6]
(Cell proliferation and apoptosis after gamma irradiation)
The intestinal epithelium has a very high regenerative capacity in, for example, humans and mice. For example, any damaged epithelial cells stop replicating and are excluded from the epithelial sheet via apoptosis. The remaining stem cells improve their proliferation rate and regenerate the crypts. As described above, if the Gsdmd gene is involved in epithelial cell homeostasis, Gsdmd null mice are considered to have altered this repair function. Therefore, Gsdmd null mice were irradiated with gamma rays to damage the intestinal epithelium, and then apoptosis of the small intestine and large intestine and cell proliferation rate were analyzed.

(Analysis of apoptosis)
First, 2 month old mice (n = 3) were irradiated with ¹³⁷ Cs-gamma rays under the conditions of an irradiation dose of 2.81 × 10 ⁻² C / Kg / min per minute and an absorbed dose of 8 Gy. After 4.5 hours of gamma irradiation, sections were taken from the small intestine (small intestine within 2 cm from the blind) and large intestine (large intestine within 2 cm from the anus) of the exposed mice. It was. These samples were analyzed for apoptosis according to the product manual using the product name ApopTag (registered trademark) Plus Peroxidase In Situ Apoptosis Detection Kit (manufactured by Chemicon). Apoptotic cell counts were scored for 30 crypts per mouse, and the number of apoptotic cells per crypt was determined by counting Apo-Tag positive cells per crypt where complete structure was seen. As a control, the same analysis was performed for wild-type mice.

  These results are shown in FIG. 10 and FIG. FIG. 10 shows the result of apoptosis analysis for the small intestine, and FIG. 11 shows the result of apoptosis analysis for the large intestine. In both figures, A shows apoptotic cells / crypts (mean ± SD) of the small intestine or large intestine of Gsdmd null mice and wild type mice, and B is a photograph of the small intestine or large intestine of Gsdmd null mice (lower left), C Is a photograph of the small or large intestine of a wild type mouse (lower right). In both figures B and C, arrows indicate apoptotic cells (magnification 400 times). For the small intestine, as shown in FIG. 10A, wild type mice showed an average of 2.77 apoptotic cells / crypts after gamma irradiation, and Gsdmd null mice showed an average of 2.33 apoptotic cells / crypts. As for the large intestine, as shown in FIG. 11A, wild-type mice showed an average of 2.77 apoptotic cells / crypts after gamma irradiation, and Gsdmd null mice showed an average of 2.41 apoptotic cells / crypts. It was. Furthermore, as for the distribution of apoptotic cells, as a result of comparing FIG. 10B and FIG. 11B (Gsdmd null mouse) with FIG. 10C and FIG. 11C (wild type mouse), no difference was observed. This result suggests that the Gsdmd gene as well as the gene product does not have a function related to apoptosis.

(Analysis of cell proliferation)
First, 2 month old mice (n = 3) were irradiated with ¹³⁷ Cs-gamma rays under the conditions of an irradiation dose of 2.81 × 10 ⁻² C / Kg / min and an absorbed dose of 10 Gy per minute. The irradiation of gamma rays was carried out until 3.5 days when cell growth peaks in the small and large intestines of healthy mice. BrdU was injected into Gsdmd null mice and wild-type mice after exposure as in “Analysis of cell proliferation” in Example 4 to detect BrdU-positive cells. As a control, the same analysis was performed for wild-type mice.

  These results are shown in FIG. 12 and FIG. FIG. 12 shows the results of cell proliferation analysis for the small intestine, and FIG. 13 shows the results of cell proliferation analysis for the large intestine. In both figures, A shows the small intestine or large intestine BrdU positive cells / crypts (mean ± SD) of Gsdmd null mice and wild type mice, B is a photograph of the small intestine or large intestine of Gsdmd null mice, and C is It is a photograph of the small intestine or large intestine of a wild type mouse (magnification rate 400 times). In both figures, the brown part is a BrdU positive proliferating cell. For the small intestine, as shown in FIG. 12A, wild-type mice showed an average of 20.49 BrdU positive cells / crypts after gamma irradiation, whereas Gsdmd null mice averaged 15.72 BrdU positive cells / negative. The pits and very small values. As for the large intestine, as shown in FIG. 13A, wild type mice showed an average of 3.58 BrdU positive cells / crypts after gamma irradiation, whereas Gsdmd null mice showed almost half of the wild type. A very small value of an average of 1.86 BrdU positive cells / crypts was shown. Analyzed values in the small and large intestines showed statistically significant differences between wild type and homozygotes (P <0.05). Furthermore, when the frequency distribution of the number of BrdU positive cells in the homozygote and the wild type was represented by a bar diagram, the difference was clear as shown in FIG. 14 and FIG. FIG. 14 shows the results of the small intestine, and FIG. 15 shows the results of the large intestine. Thus, Gsdmd null mice were found to have defects in cell proliferation and tissue recovery after intestinal epithelium was damaged. This result also suggested that the Gsdmd gene and its gene product have a function in homeostasis.

  According to the model mouse of the present invention, since the endogenous Gsdmd gene on the chromosome is knocked out, for example, functional analysis of Gsdmd and Gsdmd genes in intestinal epithelial cells can be performed. In addition, it has been clarified that the model mouse of the present invention exhibits a trait that decreases the intestinal cell proliferative function when, for example, the intestine is damaged. Therefore, by using the model mouse of the present invention in which the intestine is further damaged, it is possible to screen for a drug that restores the cell proliferation function. In addition, since the cell proliferation function in the tissue after injury is a function responsible for homeostasis, such a model mouse of the present invention is an extremely useful tool in elucidating homeostasis in the intestine.

Claims (15)

A model mouse in which the gene is knocked out,
A model mouse, wherein the mouse is a homozygote having a Gsdmd gene knocked out on a chromosome.   The model mouse according to claim 1, which is a homozygote in which exon 2 of the Gsdmd gene is deleted.   The model mouse according to claim 1, which is a homozygote in which exon 1 and exon 2 of the Gsdmd gene are deleted.   The model mouse according to claim 1, wherein the trait of the model mouse is a trait that reduces the proliferative function of intestinal cells when the intestine is damaged. The model mouse according to claim 1, which is produced by the following steps (A) to (H).
(A) Step of preparing a targeting vector having a fragment containing the genomic region of the Gsdmd gene in mouse genomic DNA (B) Introducing the targeting vector into mouse embryonic stem cells, and homologous recombination between mouse genomic DNA and the targeting vector (C) A step of selecting an embryonic stem cell in which the Gsdmd gene is knocked out by the homologous recombination (D) A step of producing a chimeric embryo cell from the embryonic stem cell obtained in the step (C) (E) The chimera Step of transplanting embryonic cells into temporary parent mice (F) Step of selecting chimeric mice born from the temporary parent mice and knocked out of the Gsdmd gene (G) Step of mating the chimeric mice and selecting heterozygous mice (H) Crossing the heterozygous mice to produce Gsdmd Step gene to select a knocked-out homozygous mice   In the step (A), the targeting vector has a cassette containing at least two Cre recombinase recognition sequences, and in the cassette, both ends of the fragment containing exon 2 of the Gsdmd gene are the recognition sequences of the Cre recombinase. The model mouse according to claim 5, which is sandwiched. The model mouse according to claim 5, further comprising the following step (I) for damaging the intestine after the step (H).
(I) A step of applying at least one selected from the group consisting of gamma irradiation, drug exposure, and bacterial infection to the knockout mouse A method for producing a model mouse in which a gene is knocked out,
The model mouse is the model mouse according to claim 1, and includes the following steps (A) to (H):
(A) Step of preparing a targeting vector having a fragment containing the genomic region of the Gsdmd gene in mouse genomic DNA (B) Introducing the targeting vector into mouse embryonic stem cells, and homologous recombination between mouse genomic DNA and the targeting vector (C) A step of selecting an embryonic stem cell in which the Gsdmd gene is knocked out by the homologous recombination (D) A step of producing a chimeric embryo cell from the embryonic stem cell obtained in the step (C) (E) The chimera Step of transplanting embryonic cells into temporary parent mice (F) Step of selecting chimeric mice born from the temporary parent mice and knocked out of the Gsdmd gene (G) Step of mating the chimeric mice and selecting heterozygous mice (H) Crossing the heterozygous mice to produce Gsdmd Step gene to select a knocked-out homozygous mice   In the step (A), the targeting vector has a cassette containing at least two Cre recombinase recognition sequences, and in the cassette, both ends of the fragment containing exon 2 of the Gsdmd gene are the recognition sequences of the Cre recombinase. The method for producing a model mouse according to claim 8, which is sandwiched.   The model mouse production method according to claim 8, wherein, in the step (A), the fragment containing exon 2 further contains exon 1. The method for producing a model mouse according to claim 8, further comprising the following step (I) for damaging the intestine after the step (H).
(I) A step of applying at least one selected from the group consisting of gamma irradiation, drug exposure, and bacterial infection to the knockout mouse   The method for producing a model mouse according to claim 9, wherein in the targeting vector, the recognition sequence of the Cre recombinase is a loxP sequence.   The method for producing a model mouse according to claim 8, wherein the targeting vector is a vector further comprising a selection marker. A method of screening for a drug that restores the cell proliferative function of damaged cells,
A screening method comprising the following steps (a) to (c):
(A) a step of preparing the model mouse according to claim 1 damaged in the intestine (b) a step of administering a candidate substance to the model mouse (c) a cell proliferating function in the intestine damaged by the model mouse For selecting candidate substances with improved chemistry as target drugs   The step (a) is a step of applying at least one selected from the group consisting of γ-irradiation, drug exposure, and bacterial infection to the model mouse according to claim 1. Screening method.

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