WO2002033054A1 - POLYβ2 KNOCKOUT ANIMALS - Google Patents

POLYβ2 KNOCKOUT ANIMALS Download PDF

Info

Publication number
WO2002033054A1
WO2002033054A1 PCT/JP2001/009204 JP0109204W WO0233054A1 WO 2002033054 A1 WO2002033054 A1 WO 2002033054A1 JP 0109204 W JP0109204 W JP 0109204W WO 0233054 A1 WO0233054 A1 WO 0233054A1
Authority
WO
WIPO (PCT)
Prior art keywords
poly
gene
mice
cells
mouse
Prior art date
Application number
PCT/JP2001/009204
Other languages
French (fr)
Japanese (ja)
Inventor
Kyoji Ikeda
Noboru Motoyama
Hiroyuki Takai
Yosuke Kobayashi
Miho Watanabe
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chugai Seiyaku Kabushiki Kaisha filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to JP2002536424A priority Critical patent/JPWO2002033054A1/en
Priority to AU2001295982A priority patent/AU2001295982A1/en
Publication of WO2002033054A1 publication Critical patent/WO2002033054A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to cells and rodents in which the poly-2 gene has been knocked out, and to uses thereof.
  • the DNA polymerase-2 (poly-2) gene contains a BRCT motif at the N-terminus and has a region at the C-terminus that shows about 30% homology with DNA polymerase (Nucleic Acids Research , 28, 3684-3693, (2000) , Journal of Molecular Biology, 301, 851-867, (2000), 'GenBank AF176098, DDBJ Keio 1019, EMBL AF176097) o, however, not been elucidated with respect to its function However, an experimental system useful for elucidating its function is required.
  • the poly-2 gene pair is inactivated and there is a genetically modified animal that has no poly52 activity, a cell line established from the genetically modified animal (genetically modified mouse-derived cell line), or an ES cell line, the individual This makes it possible to elucidate the physiological functions at the level of cells and at the cell level, and it can be used for research on therapeutics for diseases related to the gene. Disclosure of the invention
  • the present invention provides a genetically modified animal in which poly-2 gene expression is artificially suppressed, a cell line established from the genetically modified animal, a gene-modified ES cell line, and uses thereof.
  • the purpose is to:
  • the present inventors have conducted intensive studies in order to solve the above problems, and as a result, established ES cells in which one of the loci was inactivated by using gene targeting, and used the cells to develop ES cells.
  • Mera mice were produced, and by crossing using the chimeric mice, a genetically modified animal in which one or both of the poly; 52 gene pair was inactivated was successfully produced.
  • Genetically modified animals in which both gene pairs of the Poly ⁇ 2 gene have been inactivated include hydrocephalus, poor growth, visceral inversion, poor sperm maturation, cilia structure in the pilus of ventricular ependymal cells and tracheal epithelial cells It was characterized by abnormalities (dynein inner arm defect). Therefore, ES cells and animals in which the poly / 52 gene has been inactivated are effective in elucidating these diseases and developing therapeutic agents.
  • the present invention relates to a genetically modified animal and an ES cell line in which the expression of the poly ⁇ 2 gene is artificially suppressed, a cell line established from the genetically modified animal, and the use thereof.
  • the genetically modified ES cells and genetically modified animals of the present invention are characterized in that poly-2 gene expression is artificially suppressed.
  • the mouse poly /? 2 gene consisted of eight exons and was encoded on chromosome 19 ( Figure 1). In adult mice, poly 2 mRNA was expressed in almost all tissues, but the highest expression was observed in testis. poly 22 was localized in the nucleus as a protein with a molecular weight of about 60 kDa.
  • Figure 2 shows a comparison of the mouse and human poly-2 gene sequences.
  • the target animal for the modification of the poly52 gene is preferably a rodent such as a mouse, rat or hamster, and among them, a mouse is particularly preferable.
  • ES cells targeted for poly-2 gene modification are also preferably derived from rodents, and are particularly preferably derived from mice.
  • the expression of the poly-2 gene is suppressed means that the expression of the poly-2 gene is completely suppressed, or that the expression of only one gene of the gene pair is suppressed. It is intended to include the case where it is imperfectly suppressed, such as the case where In the present invention, it is preferable that the expression of the poly-2 gene is specifically suppressed.
  • a method of deleting the poly ⁇ 2 gene or a part of the expression control region of the poly ⁇ 2 gene may be used.
  • Examples of the method include a method for deleting the gene, and a method for inactivating the poly-2 gene by inserting a foreign gene into one or both of the gene pairs of the poly-2 gene is preferable. That is, in a preferred embodiment of the present invention, the genetically modified ES cell and the genetically modified animal are characterized in that a foreign gene has been inserted into one or both of the poly / -2 gene pair.
  • the genetically modified mouse of the present invention can be prepared as follows. First, DNA containing the exon portion of the poly-2 gene is isolated from a mouse, and an appropriate marker gene is inserted into this DNA fragment to construct a targeting vector. This evening targeting vector is introduced into a mouse ES cell line by an electroporation method or the like, and a cell line in which homologous recombination has occurred is selected.
  • a marker gene to be inserted an antibiotic resistance gene such as a neomycin resistance gene is preferable.
  • a cell line in which homologous recombination has occurred can be selected only by culturing in a medium containing the antibiotic.
  • the thymidine kinase gene can be linked in the evening. You. As a result, cell lines that have undergone heterologous recombination can be excluded. In addition, a diphtheria toxin A fragment (DT-A) gene or the like can be linked to the getter vector in the evening. As a result, cell lines that have undergone heterologous recombination can be excluded. In addition, a homologous recombinant can be assayed by PCR and Southern blot to efficiently obtain a cell line in which one of the gene pairs of the poly52 gene has been inactivated.
  • DT-A diphtheria toxin A fragment
  • a cell line in which homologous recombination has occurred it is preferable to create a chimera using a plurality of clones, since there is a risk of unknown gene disruption due to gene insertion in addition to the homologous recombination site.
  • the obtained ES cell line is injected into mouse blastocysts to obtain chimeric mice.
  • a mouse in which one of the gene pairs of the poly-2 gene has been inactivated can be obtained.
  • a mouse in which both gene pairs of the poly52 gene have been inactivated can be obtained.
  • Gene modification can also be performed in animals other than mice, in which ES cells have been established, by the same method.
  • An ES cell line in which both the poly-2 gene pair has been inactivated can also be obtained by the following method. That is, by culturing an ES cell line in which one of the gene pairs has been inactivated in a medium containing a high concentration of antibiotics, a cell line in which the other of the gene pair has also been inactivated, that is, the poly-2 gene, An ES cell line in which both gene pairs have been inactivated can be obtained. Alternatively, it can also be prepared by selecting an ES cell line in which one of the gene pairs has been inactivated, introducing the gettering vector again into this cell line, and selecting a cell line in which homologous recombination has occurred. it can. It is preferable to use a marker gene to be inserted into the evening-getting vector, which is different from the marker gene described above.
  • a known method can be used.
  • the method of primary culture of fetal cells can be used (Shinsei Kagaku Jikken Koza, Vol. 18, p. 125-129, Tokyo Chemical Dojin, and Ma Mouse embryo operation manual, pp. 262-264, Modern Publishing).
  • mice in which both gene pairs were inactivated were considered to have hydrocephalus. In addition, some mice exhibited visceral inversion. Furthermore, in female mice, an increase in immature sperm cells and a decrease in mature sperm cells were observed. In addition, dynein inner arm deficiency was observed in fimbriae of ependymal cells and tracheal epithelial cells. No abnormalities were found in the differentiation of T and B lymphocytes in the thymus and spleen.
  • mice in which both gene pairs of the poly-2 gene were inactivated showed hydrocephalus, poor growth, visceral inversion, poor sperm maturation, fimbriae of ependymal cells and tracheal epithelial cells.
  • mice in which both gene pairs of the poly-2 gene were inactivated showed hydrocephalus, poor growth, visceral inversion, poor sperm maturation, fimbriae of ependymal cells and tracheal epithelial cells.
  • the poly-2 gene is thought to be involved in hydrocephalus, poor growth, visceral inversion, poor sperm maturation, dynein inner arm deficiency in fimbria of ventricular ependymal cells and tracheal epithelial cells.
  • poly-2 has immotile cilia syndrome, such as total visceral inversion with bronchiectasis and chronic sinusitis, impaired ciliary movement, impaired ciliary mucus transport in respiratory airway epithelium, etc. Involvement in the characteristic Kartagener's syndrome is suggested.
  • the genetically modified animal of the present invention can be used not only for analyzing the detailed function of the polyZ gene, but also for hydrocephalus, poor growth, visceral position, It can be used to elucidate diseases such as poor sperm maturation and i-painted otile cilia syndrome. Furthermore, it can be used for screening of therapeutic agents for those diseases.
  • poly-2 gene and protein can be used as therapeutics for the above diseases, etc.
  • poly ⁇ 2 gene is expected to be used for the treatment of the above diseases by gene therapy. You. Brief description of the drawings ''
  • FIG. 1 shows the exon 'intron structure of the mouse poly / 52 gene.
  • A shows the overall image of the exon-intron structure
  • B shows the nucleotide sequence of the boundary region between the exon and the intron.
  • FIG. 2 shows a comparison of the nucleotide sequences of the mouse and human poly / 52 genes.
  • FIG. 4 is a photograph showing a section of the brain of a poly52 + / + mouse and a poly / 52 ⁇ / ⁇ mouse.
  • Embryo 14.5 is a photograph showing a cross-section of the brain of poly /? 2 + / + mice (left) and poly 52-/-mice (right).
  • Figure 5 is a photograph showing a serial sagittal section of the brain.
  • FIG. 4g The ventricular ependymal cells of the poly? 2 + / + mouse make up several layers of cells surrounding the ventricle (Fig. 4g, h), but in the brain of the poly52-/-mouse (right). Hyperplasia of ependymal cells in the third ventricle (3V) and middle cerebral aqueduct (Aq) is observed, and the layer structure is broken (Fig. 4c-f). As can be seen from the enlarged photograph inserted in c, at the hyperplastic site of the ependymal cells, there are mononuclear cells with a spherical, well-defined nucleus and mononuclear cells with eosinphilic cytoplasm (Fig. 4c).
  • FIG. 6 is a photograph showing the result of examining the localization of a transcript (mRNA) of the poly32 gene in the brain and testis by in situ hybridization.
  • mRNA transcript
  • a, b Photographs of a sperm obtained from the epididymis tail observed with a phase contrast microscope. Compared to the shape of spermatozoa of 9-week-old poly? 2 + / + mice (a), the spermatozoa of poly /? 2-/-mice (b) have short tails, those without tails, Malformed spermatozoa such as those with a circular head were seen.
  • c-h are photographs showing the results of pathological analysis of testis.
  • the poly? 2-/-mouse testis (d) has the same tubule structure as the poly / 52 + ⁇ mouse testis (c).
  • 3 is a photograph showing the results of pathological analysis of the epididymis body part. .
  • Poly 32 + / + mice (i) show mature sperm cells, whereas poly /? 2-/-mice (j) have few mature sperm cells, immature sperm cells and eosinophils Many substances were found.
  • FIG. 8 shows the survival curve of poly 2 ⁇ / ⁇ mice.
  • FIG. 4 presents photographs showing the results of Northern blot analysis of the expression of poly ⁇ Z gene transcript (mRNA) in testis of 'c. poly? 2-/-mice. EF-1 was used as a control. .
  • FIG. 10 is an anatomical photograph showing the internal organ arrangement of poly-2 ⁇ / ⁇ and poly-2 + / + mice. Ribs and liver have been resected for better visibility of internal organs. The internal organs of poly ⁇ 2 ⁇ / ⁇ mice are mirror images of the internal organs of poly 2 + / + mice. c In the figure, Ap is Apex (apical), Fs is Forestomach (foregoma), and Sp is Spleen. (Spleen).
  • FIG. 11 is a photograph of the pilus structure of a poly-2 /-mouse observed with an electron microscope.
  • the upper part shows the cross section of the fimbria of the ependymal cells of the ventricle, and the lower part shows the cross section of the pili of the tracheal epithelial cells.
  • 2- /-mice dynein inner arm deficiency is found in the pili of ependymal cells (upper right) and the pili of tracheal epithelial cells (lower right).
  • Figure 12 is a schematic diagram of two dynein arms (outer arm and inner arm) of a double microtubule.
  • the mouse poly52 genomic gene was cloned.
  • plaque hybridization was performed using mouse poly-2c DNA as a probe, and a genomic DNA fragment of approximately 20kb containing poly / -2 gene exon 1 was isolated. The site was mapped.
  • exon 1 to exon 6 of the poly-2 gene with the neomycin resistance gene expression cassette by homologous recombination include the gene fragment containing the 5 'side sequence of exon 1 and exons 7 and 8 at both ends of the neomycin resistance gene expression cassette
  • the homologous region was arranged by ligating the gene fragments, and the tk expression cassette was further ligated to the 3 'downstream to create a homologous recombination vector (FIG. 9a, upper panel).
  • the poly-2 locus has a restriction enzyme Xbal recognition site (X) upstream of exon 1 and a restriction enzyme Spel recognition site (Sp) between exons 3 and 4 (middle of Fig. 9a).
  • the homologous recombination vector was introduced into mouse ES cell E14 (Hopper, M. et al .: Nature, 326, 292-295, 1987) by electroporation, followed by G418 and ganciclovir. Selection culture was performed. The obtained G418Z ganciclovir-resistant colonies were tested for homologous recombinants by PCR and Southern blotting, and four cell lines in which the mutant poly 2 gene was inserted into one of the poly / -2 gene pairs were obtained.
  • the resulting clones were confirmed by Southern analysis for insertion of the mutant gene (FIG. 9b).
  • the mutant gene was not inserted, only the llkb band was detected, whereas the cell lines 96, 97, and 110 obtained by introducing the vector for homologous recombination and selecting the drug were used.
  • a llkb band derived from the normal poly-2 locus and a 9.2-kb band derived from the mutant poly / -2 locus were detected.
  • ES cells in which one of the poly ⁇ 2 genes was inactivated were injected into blastocysts derived from C57BL / 6 mice to obtain chimeric mice.
  • a chimeric mouse prepared using ES cells in which one of the poly-2 gene pairs has been inactivated is crossed with C57BL / 6 mice, and one of the poly-2 genes pairs consists of inactivated cells
  • a mouse (hereinafter, referred to as "poly? 2 +/- mouse") was obtained.
  • poly? 2-/-mice mice in which both poly ⁇ 2 gene pairs were inactivated
  • mice with marked lateral ventricular dilation show apoptosis in ependymal cells around the ventricle. Such apoptosis is not observed around the ventricle before ventricular dilation.
  • Ventricular dilatation is seen in the lateral and third ventricles, but not in the fourth and middle ventricles.
  • hyperplasia of ependymal cells in the third ventricle and the middle cerebral aqueduct is observed from day 14.5 onward (Fig. 4c-e).
  • the ventricular ependymal cells comprise several layers of cells that surround the ventricle (Fig. 5g, h).
  • hyperplasia of the ependymal cells of the third ventricle and the middle cerebral aqueduct was observed, and the layer structure was disrupted (Fig. 5 af).
  • mononuclear cells with a spherical and well-defined nucleus and mononuclear cells with eosinphilic cytoplasm are observed (Fig. 5c).
  • Electron microscopy of ependymal cells showing hydrocephalus reveals multilayering of ependymal cells and disruption of the microvilli margin (Fig. 4f).
  • mice in which both poly-2 genes are inactivated is due to cerebrospinal fluid production and / or reabsorption due to occlusion of the cerebrospinal fluid circulation and disruption of ependymal microvillous margins. It is considered abnormal.
  • Poly ⁇ 2-/-mice with hydrocephalus show growth retardation compared to wild-type mice in which the poly ⁇ 2 gene is not inactivated (hereinafter referred to as "poly52 + / + mice”). 3 a). On days 14 and 21 after birth, poly? 2-/-mice weighed poly 2 + / + About 60-80% of mice.
  • poly ⁇ 2-/-mice begin to die at 3 weeks of age and have a 50% survival rate by 5 weeks of age (Figure 8).
  • the dead poly 2 ⁇ / ⁇ mice are mostly individuals with hydrocephalus, and the poly 52 ⁇ / ⁇ mice surviving beyond 5 weeks of age are apparently identical to normal mice.
  • poly-2 mRNA The localization of the transcript (mRNA) of the poly52 gene was examined by in situ and hybridization.
  • poly-2 mRNA is localized in cells around the ventricle of the fetal brain of wild-type mice and in adult ependymal ependymal cells and choroid plexus.In poly52-/-mice, poly-2 mRNA is expressed in those cells. No localization is observed ( Figures 6a-c).
  • the localization of the transcript (mRNA) of the poly /? 2 gene was examined by in situ hybridization. Poly-2mMA is localized in the testes of wild-type mice. In poly / 52-/-mice, no localization of poly-2 mRNA in testis was observed (Fig. 6d). .
  • RNA poly-2 transcripts
  • Fig. 9c The expression of poly-2 transcripts (mRNA) in testis of poly-2 /-mice was analyzed by Northern blot analysis (Fig. 9c). In poly? 2 + / + mice and (poly? 2 +/- mice, the expression of poly /? 2 gene mRNA is observed, but in poly /? 2-/-mice, the expression of poly? 2 gene is observed.
  • the lower panel in Fig. 9c shows the expression of EF-1 cells, which demonstrates the presence of the RNA sample in each lane.
  • Dyskinesia due to ciliary structural abnormalities similar to those found in cilia of tracheal epithelial cells and ventricular ependymal cells also occur in the cilia of nasal mucociliary cells that make up the nasal mucosa It is presumed that rhinitis has developed.
  • ES cells and mice in which poly-2 gene expression is artificially suppressed wherein the mice have hydrocephalus, poor growth, visceral inversion, poor sperm maturation, ventricular ependymal cells and It was found to be characterized by abnormal ciliary structure (dynein inner arm defect) in the pili of tracheal epithelial cells. Therefore, if the genetically modified ES cells, genetically modified animals, or cell lines established from the animals of the present invention are used, the function of the poly /? 2 gene can be analyzed, as well as hydrocephalus, poor growth, visceral inversion, and sperm.
  • poly-2 gene and protein can be used as therapeutic agents for the above-mentioned diseases and the like.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Genetically modified animals, in which one or both of the gene pair of polyß gene are inactivated, are successfully constructed by establishing ES cells in which one of the gene loci is inactivated by using the gene targeting method, constructing chimeric mice by using these cells, and mating the thus obtained chimeric mice. These knockout mice are characterized by suffering from hypocephalus, hypoplasis, visceral inversion, spermatozoal maturity failure and cilia structural failure in pili in ependymal cells and tracheal epithelial cells (dynein inner-arm missing).

Description

poly/? 2遺伝子欠損動物 技術分野  poly /? 2 gene deficient animal
本発明は、 poly ?2遺伝子がノックアウトされた細胞およびげつ歯類動物、 並び にそれらの利用に関する。 背景技術  The present invention relates to cells and rodents in which the poly-2 gene has been knocked out, and to uses thereof. Background art
DNAポリメラーゼ ? 2 (poly ?2) 遺伝子は N末端に BRCTモチーフを含み、 C末端側 に DNAポリメラ一ゼ ?と 30%程度の相同性を示す領域を有している遺伝子である (Nucleic Acids Research, 28， 3684-3693, (2000), Journal of Molecular Biology, 301, 851-867, (2000),' GenBank AF176098, DDBJ應 1019， EMBL AF176097)o しかしながら、 その機能に関しては未だ解明されておらず、 その機能の解明に有 用な実験系が求められている。 The DNA polymerase-2 (poly-2) gene contains a BRCT motif at the N-terminus and has a region at the C-terminus that shows about 30% homology with DNA polymerase (Nucleic Acids Research , 28, 3684-3693, (2000) , Journal of Molecular Biology, 301, 851-867, (2000), 'GenBank AF176098, DDBJ Keio 1019, EMBL AF176097) o, however, not been elucidated with respect to its function However, an experimental system useful for elucidating its function is required.
poly ?2遺伝子対が不活性化され、 poly 52の活性を全く持たない遺伝子改変動物、 遺伝子改変動物から樹立された細胞株（遺伝子改変マウス由来細胞株）、 あるいは ES細胞株があれば、個体レベルおよび細胞レベルでの生理機能の解明が可能となり、 該遺伝子に関連した疾患の治療薬などの研究に利用することが可能となる。 発明の開示  If the poly-2 gene pair is inactivated and there is a genetically modified animal that has no poly52 activity, a cell line established from the genetically modified animal (genetically modified mouse-derived cell line), or an ES cell line, the individual This makes it possible to elucidate the physiological functions at the level of cells and at the cell level, and it can be used for research on therapeutics for diseases related to the gene. Disclosure of the invention
そこで、 本発明は、 poly ?2遺伝子の発現が人為的に抑制された遺伝子改変動物 および該遺伝子改変動物から樹立された細胞株、及び遣伝子改変 ES細胞株、並びに それらの用途を提供することを目的とする。  Accordingly, the present invention provides a genetically modified animal in which poly-2 gene expression is artificially suppressed, a cell line established from the genetically modified animal, a gene-modified ES cell line, and uses thereof. The purpose is to:
本発明者らは、上記課題を解決する為に鋭意検討を重ねた結果、 ジーン夕ーゲテ ィングを用い片方の遺伝子座を不活性化した ES細胞を樹立し、その細胞を用いてキ メラマウスを作成し、 該キメラマウスを用いた交配により、 poly；52遺伝子の遺伝 子対の一方または双方が不活性化された遺伝子改変動物を作成することに成功し た。 Poly^2遺伝子の遺伝子対の双方が不活性化された遺伝子改変動物は、水頭症、 発育不良、 内臓逆位、 精子の成熟不良、 脳室上衣細胞および気管上皮細胞の線毛に おける繊毛構造異常 （ダイニン内腕欠損） を特徴としていた。 従って、 poly/52遺 伝子が不活性化された ES細胞や動物は、これら疾患の解明や治療薬などの開発など に有効である。 The present inventors have conducted intensive studies in order to solve the above problems, and as a result, established ES cells in which one of the loci was inactivated by using gene targeting, and used the cells to develop ES cells. Mera mice were produced, and by crossing using the chimeric mice, a genetically modified animal in which one or both of the poly; 52 gene pair was inactivated was successfully produced. Genetically modified animals in which both gene pairs of the Poly ^ 2 gene have been inactivated include hydrocephalus, poor growth, visceral inversion, poor sperm maturation, cilia structure in the pilus of ventricular ependymal cells and tracheal epithelial cells It was characterized by abnormalities (dynein inner arm defect). Therefore, ES cells and animals in which the poly / 52 gene has been inactivated are effective in elucidating these diseases and developing therapeutic agents.
即ち、 本発明は、 poly^2遺伝子の発現が人為的に抑制されている遺伝子改変動 物および ES細胞株、および当該遺伝子改変動物から樹立された細胞株、並びにそれ らの利用に関し、 より詳しくは、  That is, the present invention relates to a genetically modified animal and an ES cell line in which the expression of the poly ^ 2 gene is artificially suppressed, a cell line established from the genetically modified animal, and the use thereof. Is
( 1 ) poly/? 2遺伝子の発現が人為的に抑制されていることを特徴とするげつ歯 類 ES細胞、  (1) a rodent ES cell, wherein the expression of the poly /? 2 gene is artificially suppressed,
( 2 ) poly ?2遺伝子の遺伝子対の一方または双方に外来遺伝子が挿入されてい ることを特徴とするげつ歯類 ES細胞、  (2) a rodent ES cell comprising a foreign gene inserted into one or both of the poly-2 gene pairs,
( 3 ) げっ歯類 ES細胞がマウス ES細胞である （ 1 ) または （2 ) 記載の ES細胞、 (3) The ES cell according to (1) or (2), wherein the rodent ES cell is a mouse ES cell.
( 4 ) poly^ 2遺伝子の発現が人為的に抑制されていることを特徴とする遺伝子 改変げつ歯類動物、 (4) a genetically modified rodent, wherein expression of the poly ^ 2 gene is artificially suppressed;
( 5 ) poly 52遺伝子の造伝子対の一方または双方に外来遺伝子が挿入されてい ることを特徴とするげつ歯類動物、  (5) a rodent, wherein a foreign gene is inserted into one or both of the gene pairs of the poly52 gene;
( 6 ) げっ歯類動物がマウスである （4 ) または （5 ) 記載の動物、 および (6) the animal according to (4) or (5), wherein the rodent is a mouse; and
( 7 ) ( 4 ) 〜 （6 ) のいずれかに記載の動物から樹立された細胞株、 を提供す るものである。 (7) A cell line established from the animal according to any one of (4) to (6).
本発明の遺伝子改変 ES細胞および遺伝子改変動物は、 poly ?2遺伝子の発現が人 為的に抑制されていることを特徴とする。マウス poly/? 2遺伝子は 8つのェクソンか らなり、 19番染色体上にコードされていた（図 1 )。成体マウスにおいて po ly 2mRNA はほぼすベての組織に発現しているが、 精巣で最も発現が高く認められた。 poly ?2は分子量約 60kDaの蛋白質として核に局在していた。 マウスとヒトの poly ?2遺 伝子の塩基配列の比較を図 2に示す。 The genetically modified ES cells and genetically modified animals of the present invention are characterized in that poly-2 gene expression is artificially suppressed. The mouse poly /? 2 gene consisted of eight exons and was encoded on chromosome 19 (Figure 1). In adult mice, poly 2 mRNA was expressed in almost all tissues, but the highest expression was observed in testis. poly 22 was localized in the nucleus as a protein with a molecular weight of about 60 kDa. Figure 2 shows a comparison of the mouse and human poly-2 gene sequences.
本発明において poly 52遺伝子の改変の対象となる動物は、 好ましくはマウス、 ラヅト、 ハムスターなどのげつ歯類であり、 その中でも特にマウスが好ましい。本 発明において poly ?2遺伝子の改変の対象となる ES細胞もまた、 げっ歯類に由来す るものが好ましく、 特にマウス由来が好ましい。  In the present invention, the target animal for the modification of the poly52 gene is preferably a rodent such as a mouse, rat or hamster, and among them, a mouse is particularly preferable. In the present invention, ES cells targeted for poly-2 gene modification are also preferably derived from rodents, and are particularly preferably derived from mice.
本発明において「poly ?2遺伝子の発現が抑制されている」 とは、 poly ?2遺伝子 の発現が完全に抑制されている場合の他、遺伝子対の一方の遺伝子の発現のみが抑 制されている場合のように、不完全に抑制されている場合も含む意である。本発明 においては、 poly ?2遺伝子の発現が特異的に抑制されていることが好ましい。 本発明の遺伝子改変 ES細胞および遺伝子改変動物において、 poly 52遺伝子の発 現を人為的に抑制する手段としては、 poly^2遺伝子を欠損させる方法や poly^2 遺伝子の発現制御領域の一部を欠損させる方法等を例示することができるが、好ま しくは poly ?2遺伝子の遺伝子対の一方または双方に外来遺伝子を挿入することに より poly ?2遺伝子を不活性化する方法である。 即ち、 本発明の好ましい態様にお いて、 遺伝子改変 ES細胞および遺伝子改変動物は、 poly/? 2遺伝子の遺伝子対の一 方または双方に外来遺伝子が挿入されていることを特徴とする。  In the present invention, "the expression of the poly-2 gene is suppressed" means that the expression of the poly-2 gene is completely suppressed, or that the expression of only one gene of the gene pair is suppressed. It is intended to include the case where it is imperfectly suppressed, such as the case where In the present invention, it is preferable that the expression of the poly-2 gene is specifically suppressed. As means for artificially suppressing the expression of the poly52 gene in the genetically modified ES cells and the genetically modified animals of the present invention, a method of deleting the poly ^ 2 gene or a part of the expression control region of the poly ^ 2 gene may be used. Examples of the method include a method for deleting the gene, and a method for inactivating the poly-2 gene by inserting a foreign gene into one or both of the gene pairs of the poly-2 gene is preferable. That is, in a preferred embodiment of the present invention, the genetically modified ES cell and the genetically modified animal are characterized in that a foreign gene has been inserted into one or both of the poly / -2 gene pair.
本発明の遺伝子改変マウスは、 以下のようにして作成することができる。 まず、 マウスから poly ?2遺伝子のェクソン部分を含む DNAを単離し、 この DNA断片に適当 なマーカー遺伝子を揷入し、 夕一ゲヅティングベクターを構築する。この夕ーゲヅ ティングベクターをエレクトロポーレーシヨン法などによりマウス ES細胞株に導 入し、相同組み換えを生じた細胞株を選抜する。挿入するマーカー遺伝子としては、 ネオマイシン耐性遺伝子などの抗生物質耐性遺伝子が好ましい。抗生物質耐性遺伝 子を挿入した場合には、抗生物質を含む培地で培養するだけで相同組み換えを生じ た細胞株を選抜することができる。また、 より効率的な選抜を行うためには夕ーゲ ッティングぺク夕一にチミジンキナーゼ遺伝子などを結合させておくこともでき る。これにより、非相同組み換えを起こした細胞株を排除することができる。また、 夕一ゲッティングベクタ一にジフテリアトキシン Aフラグメント（DT- A)遺伝子など を結合させておくこともできる。これにより、非相同的な組換えを起こした細胞株 を排除することができる。 また、 PCRおよびサザンブロットにより相同組み換え体 の検定を行い、 poly 52遺伝子の遺伝子対の一方が不活性化された細胞株を効率的 に得ることもできる。 The genetically modified mouse of the present invention can be prepared as follows. First, DNA containing the exon portion of the poly-2 gene is isolated from a mouse, and an appropriate marker gene is inserted into this DNA fragment to construct a targeting vector. This evening targeting vector is introduced into a mouse ES cell line by an electroporation method or the like, and a cell line in which homologous recombination has occurred is selected. As the marker gene to be inserted, an antibiotic resistance gene such as a neomycin resistance gene is preferable. When an antibiotic-resistant gene is inserted, a cell line in which homologous recombination has occurred can be selected only by culturing in a medium containing the antibiotic. For more efficient selection, the thymidine kinase gene can be linked in the evening. You. As a result, cell lines that have undergone heterologous recombination can be excluded. In addition, a diphtheria toxin A fragment (DT-A) gene or the like can be linked to the getter vector in the evening. As a result, cell lines that have undergone heterologous recombination can be excluded. In addition, a homologous recombinant can be assayed by PCR and Southern blot to efficiently obtain a cell line in which one of the gene pairs of the poly52 gene has been inactivated.
相同組み換えを生じた細胞株を選抜する場合、相同組み換え箇所以外にも、遺伝 子挿入による未知の遺伝子破壊の恐れがあるので、複数のクローンを用いてキメラ 作成を行うことが好ましい。得られた ES細胞株をマウス胚盤胞にィンジェクシヨン しキメラマウスを得ることができる。 このキメラマウスを交配させることで、 poly ?2遺伝子の遺伝子対の一方を不活性化したマウスを得ることができる。 さらに、 このマウスを交配させることで、 poly 52遺伝子の遺伝子対の双方を不活性化した マウスを得ることができる。マウス以外の ES細胞が樹立された動物においても、 同 様の手法により、 遺伝子改変を行なうことができる。  When selecting a cell line in which homologous recombination has occurred, it is preferable to create a chimera using a plurality of clones, since there is a risk of unknown gene disruption due to gene insertion in addition to the homologous recombination site. The obtained ES cell line is injected into mouse blastocysts to obtain chimeric mice. By mating this chimeric mouse, a mouse in which one of the gene pairs of the poly-2 gene has been inactivated can be obtained. Furthermore, by mating this mouse, a mouse in which both gene pairs of the poly52 gene have been inactivated can be obtained. Gene modification can also be performed in animals other than mice, in which ES cells have been established, by the same method.
また、 poly ?2遺伝子の遺伝子対の双方を不活性化した ES細胞株は、 以下の方法 により取得することも可能である。 すなわち、 遺伝子対の一方を不活性化した ES 細胞株を高濃度の抗生物質を含む培地で培養することにより、遺伝子対のもう一方 も不活性化された細胞株、 即ち、 poly ?2遺伝子の遺伝子対の双方を不活性化した ES細胞株を得ることができる。 また、遺伝子対の一方を不活性化した ES細胞株を選 抜し、 この細胞株に再度夕一ゲッティングベクターを導入し、相同組換えを生じた 細胞株を選択することでも作製することができる。夕ーゲッティングベクターに揷 入するマーカ一遺伝子は、前出のマーカ一遺伝子とは異なるものを使用することが 好ましい。  An ES cell line in which both the poly-2 gene pair has been inactivated can also be obtained by the following method. That is, by culturing an ES cell line in which one of the gene pairs has been inactivated in a medium containing a high concentration of antibiotics, a cell line in which the other of the gene pair has also been inactivated, that is, the poly-2 gene, An ES cell line in which both gene pairs have been inactivated can be obtained. Alternatively, it can also be prepared by selecting an ES cell line in which one of the gene pairs has been inactivated, introducing the gettering vector again into this cell line, and selecting a cell line in which homologous recombination has occurred. it can. It is preferable to use a marker gene to be inserted into the evening-getting vector, which is different from the marker gene described above.
本発明の遺伝子改変動物由来の細胞株を樹立する方法としては、公知の方法を用 いることができる。例えば、 げっ歯類においては、 胎仔細胞の初代培養の方法を用 いることができる （新生化学実験講座、 18巻、 125頁〜 129頁東京化学同人、 及びマ ウス胚の操作マニュアル、 262頁〜 264頁、 近代出版） 。 As a method for establishing the cell line derived from the genetically modified animal of the present invention, a known method can be used. For example, in rodents, the method of primary culture of fetal cells can be used (Shinsei Kagaku Jikken Koza, Vol. 18, p. 125-129, Tokyo Chemical Dojin, and Ma Mouse embryo operation manual, pp. 262-264, Modern Publishing).
本実施例において作成された poly ?2遺伝子の遺伝子対の双方が不活性化された マウスは、通常の割合で生まれ、 出生直後は外見上の異常は観察されなかった。 し かし、 生後 2週間以降、 発育不良と頭部の膨隆が観察された。 病理解析の結果、 側 脳室、 第三脳室の著名な拡大と脳実質の減少が観察された。 このことから、 poly Mice in which both gene pairs of the poly-2 gene created in this example were inactivated were born at a normal ratio, and no apparent abnormality was observed immediately after birth. However, from 2 weeks after birth, poor growth and bulging of the head were observed. As a result of pathological analysis, remarkable enlargement of the lateral ventricle and third ventricle and a decrease in brain parenchyma were observed. From this, poly
2遺伝子の遺伝子対の双方が不活性化されたマウスは水頭症を発症していると考 えられた。 また、 内臓逆位が観察されたマウスも存在した。 さらに、 ォスマウスに 'おいては未熟な精子細胞の増加と、 成熟した精子細胞の減少が認められた。 また、 脳室上衣細胞および気管上皮細胞の線毛におけるダイニン内腕欠損が認められた。 胸腺、 脾臓における Tリンパ球および Bリンパ球の分化に異常は認められなかった。 以上の結果から、 poly ?2遺伝子の遺伝子対の双方が不活性化されたマウスは、 水 頭症、 発育不良、 内臓逆位、 精子の成熟不良、 脳室上衣細胞および気管上皮細胞の 線毛におけるダイニン内腕欠損を特徴とすることが判明した。 即ち、， poly ?2遺伝 子は水頭症、 発育不良、 内臓逆位、 精子の成熟不良、 脳室上衣細胞および気管上皮 細胞の線毛におけるダイニン内腕欠損等に関与していると考えられる。 また、 これ らの結果から poly ?2は immotile cilia症候群、 例えば気管支拡張症と慢性副鼻腔 炎を伴う全内臓逆位症、繊毛の動きの障害、 呼吸気道上皮における線毛粘液移送の 障害などを特徴とする Kartagener' s症候群への関与が示唆される。 Mice in which both gene pairs were inactivated were considered to have hydrocephalus. In addition, some mice exhibited visceral inversion. Furthermore, in female mice, an increase in immature sperm cells and a decrease in mature sperm cells were observed. In addition, dynein inner arm deficiency was observed in fimbriae of ependymal cells and tracheal epithelial cells. No abnormalities were found in the differentiation of T and B lymphocytes in the thymus and spleen. Based on the above results, mice in which both gene pairs of the poly-2 gene were inactivated showed hydrocephalus, poor growth, visceral inversion, poor sperm maturation, fimbriae of ependymal cells and tracheal epithelial cells. Was found to be characterized by a dynein inner arm defect. In other words, the poly-2 gene is thought to be involved in hydrocephalus, poor growth, visceral inversion, poor sperm maturation, dynein inner arm deficiency in fimbria of ventricular ependymal cells and tracheal epithelial cells. In addition, these results indicate that poly-2 has immotile cilia syndrome, such as total visceral inversion with bronchiectasis and chronic sinusitis, impaired ciliary movement, impaired ciliary mucus transport in respiratory airway epithelium, etc. Involvement in the characteristic Kartagener's syndrome is suggested.
従って、本発明の遺伝子改変動物、該動物から樹立した細胞株、及び ES細胞株は、 poly Z遺伝子の詳細な機能の解析に利用できるだけでなく、 例えば、 水頭症、 発 育不良、 内臓 位、 精子の成熟不良、 i画 otile cilia症候群などの疾患の解明に利 用できる。さらに、 それらの疾患の治療薬などのスクリーニングにも利用すること が可能である。 また、 poly ?2遺伝子及びタンパク質は上記疾患の治療薬などとし て利用することが可能であると考えられ、 特に、 poly^2遺伝子は、 遺伝子治療に よる上記疾患の治療への利用が期待される。 図面の簡単な説明 ' Therefore, the genetically modified animal of the present invention, the cell line established from the animal, and the ES cell line can be used not only for analyzing the detailed function of the polyZ gene, but also for hydrocephalus, poor growth, visceral position, It can be used to elucidate diseases such as poor sperm maturation and i-painted otile cilia syndrome. Furthermore, it can be used for screening of therapeutic agents for those diseases. In addition, it is thought that poly-2 gene and protein can be used as therapeutics for the above diseases, etc. In particular, poly ^ 2 gene is expected to be used for the treatment of the above diseases by gene therapy. You. Brief description of the drawings ''
図 1は、 マウス poly/52遺伝子のェクソン 'イントロン構造を示す図である。 A は、 ェクソン ·イントロン構造の全体像を示し、 Bは、 ェクソンとイントロンの境 界領域の塩基配列を示す。  FIG. 1 shows the exon 'intron structure of the mouse poly / 52 gene. A shows the overall image of the exon-intron structure, and B shows the nucleotide sequence of the boundary region between the exon and the intron.
図 2は、 マウスとヒトの poly/52遺伝子の塩基配列の比較を示す ¾である。  FIG. 2 shows a comparison of the nucleotide sequences of the mouse and human poly / 52 genes.
図 3は、  Figure 3 shows
a .生後 4週齢の、 poly ?2+/+マウス （左：ページュ） と poly/? 2 -/-マウス （右： 白） を示す写真である。  a. Photographs showing a 4-week-old poly-2 + / + mouse (left: page) and a poly-2 / 2-/-mouse (right: white).
poly ?2 -/-マウスでは頭部の膨隆が見られ、 また発育の遅れが観察される。 b . 生後 5週齢の、 poly 32+/+マウス （左） と poly/? 2 -/-マウス （右） の脳の外 部形態を示した写真である。  Poly? 2-/-mice show head bulges and delayed development. b. Photographs showing the external morphology of the brain of a poly32 + / + mouse (left) and a poly /? 2-/-mouse (right), 5 weeks old.
エーテル麻酔による安楽死の後、 頭部皮膚を切開し、 頭蓋骨を部分的に除去し、 大脳上部を露出させた。 poly ?2- /-マウス （右） では、 脳の拡張と大脳皮質の薄層 化が観察される。  After euthanasia with ether anesthesia, the skin of the head was cut open, the skull was partially removed, and the upper cerebrum was exposed. In poly? 2- /-mice (right), dilation of the brain and thinning of the cerebral cortex are observed.
c 生後 5週齢の、 poly ?2+/+マウス （左） と poly ?2- /-マウス （右） の脳の冠 状断面 （上段） と矢状断面 （下段） をしめした写真である。  c Photograph showing the coronal (upper) and sagittal (lower) sections of the brain of a 5 week old poly-2 + / + mouse (left) and poly-2 /-mouse (right). .
bと同じくエーテル麻酔による安楽死の後、頭部を剖出し、冠状断標本及び矢状 断標本を作成した。 poly ?2- /-マウス （右） では、 側脳室の拡張と大脳皮質の薄層 化が観察される。  After euthanasia under ether anesthesia as in b), the head was dissected and coronal and sagittal sections were prepared. In poly? 2- /-mice (right), dilatation of the lateral ventricle and thinning of the cerebral cortex are observed.
図 4は、 poly 52+/+マウスと poly/52- /-マウスの脳の切片標本を示した写真であ る。  FIG. 4 is a photograph showing a section of the brain of a poly52 + / + mouse and a poly / 52 − / − mouse.
a . 生後 1日の poly /52+/+マウス （左） と poly 52- /-マウス （右） の脳の横断切 片標本を示した写真である。  a. Photographs showing brain cross-sections of poly / 52 + / + mice (left) and poly52-/-mice (right) one day after birth.
poly ?2- /-マウス （右） では、 側脳室の拡張が認められる。  In poly? 2- /-mice (right), lateral ventricle dilation is observed.
b . 生後 5週の、 poly^2+/+マウス （左） と poly 52- /-マウス （右） の脳の横断 切片標本を示した写真である。 双方の poly ?2遺伝子が不活性化されたマウス （右） において脳室の拡張が認め られる。 b. Photograph showing cross-sectioned brain samples of poly ^ 2 + / + mice (left) and poly52-/-mice (right) at 5 weeks of age. Ventricular dilatation is observed in mice in which both poly-2 genes have been inactivated (right).
c . 胎生 14.5曰の、 poly/? 2+/+マウス （左） と poly 52-/-マウス （右） の脳の横 断切片標本を示した写真である。  c. Embryo 14.5 is a photograph showing a cross-section of the brain of poly /? 2 + / + mice (left) and poly 52-/-mice (right).
poly/52- /-マウス （右） において、 脳室上衣細胞の細胞分裂を伴う過形成が認め られる。  In poly / 52 − / − mice (right), hyperplasia with cell division of ependymal ependymal cells is observed.
d . 生後 1日の、 poly ?2+/+マウス （左） と poly/? 2-/-マウス （右） の脳の横断 切片標本を示した写真である。  d. Photograph showing a cross-sectional specimen of the brain of a poly-2 + / + mouse (left) and a poly / -2 /-mouse (right) one day after birth.
poly ?2-/-マウス （右） において、 脳室上衣細胞の乳頭状の細胞増殖を伴う過形 成が認められる。  In poly? 2-/-mice (right), hyperplasia with papillary cell proliferation of ventricular ependymal cells is observed.
e . 生後 5週齢の、 poly ?2+/+マウス （左） と poly/52- /-マウス （右） の脳の横 断切片標本を示した写真である。  e. Photograph showing a cross section specimen of the brain of a 5 week old poly-2 + / + mouse (left) and poly / 52-/-mouse (right).
poly ?2-/-マウス （右） において、 第三脳室上衣細胞の乳頭状の細胞増殖を伴う 過形成が認められる。  In poly? 2-/-mice (right), hyperplasia with papillary cell proliferation of third ventricular ependymal cells is observed.
f . 生後 5週齢の、 poly ?2+/+マウス （左） と poly ? 2- /-マウス （右） の脳の電 子顕微鏡解析標本を示した写真である。  f. Photo showing electron microscope analysis specimens of the brains of poly-2 + / + mice (left) and poly-2 /-mice (right) at 5 weeks of age.
poly ?2- /-マウス （右） において、 上衣細胞の多層化および微絨毛縁の崩壊が認 められる。  In poly? 2- /-mice (right), multilayering of ependymal cells and disruption of microvilli margins are observed.
図 5は、 脳の矢状断連続切片標本を示した写真である。  Figure 5 is a photograph showing a serial sagittal section of the brain.
aに示すように正中面近くの断面の連続切片 （c、 d、 e、 f) を作成した。  As shown in a, continuous sections (c, d, e, f) of the cross section near the median plane were prepared.
poly ? 2+/+マウスの脳室上衣細胞は、脳室を取り巻くように数層の細胞層を構成 しているが （図 4g、 h)、 poly 52- /-マウス （右） の脳では第三脳室 (3V)および中 脳水道（Aq)の上衣細胞の過形成が見られ、 層構造が破綻している部分が認められ る （図 4c- f)。 cに挿入した拡大写真でわかるように、上衣細胞の過形成部位では、 球形で明確な核をもつ単核細胞とェォジン好性の細胞質をもつ単核細胞が認めら れる （図 4c) 。 図 6は、脳および精巣における poly 32遺伝子の転写産物（mRNA)の局在を in situ ハイプリダイゼーシヨンにより調べた結果を示す写真である。 The ventricular ependymal cells of the poly? 2 + / + mouse make up several layers of cells surrounding the ventricle (Fig. 4g, h), but in the brain of the poly52-/-mouse (right). Hyperplasia of ependymal cells in the third ventricle (3V) and middle cerebral aqueduct (Aq) is observed, and the layer structure is broken (Fig. 4c-f). As can be seen from the enlarged photograph inserted in c, at the hyperplastic site of the ependymal cells, there are mononuclear cells with a spherical, well-defined nucleus and mononuclear cells with eosinphilic cytoplasm (Fig. 4c). FIG. 6 is a photograph showing the result of examining the localization of a transcript (mRNA) of the poly32 gene in the brain and testis by in situ hybridization.
a. 胎生 14,5日の poly 32+/+マウス （左） の脳では、 脳室周辺の細胞で poly 32 遺伝子 mRNAの局在が見られるが、 poly ?2- /-マウス （右） の脳ではそれらは見られ ない。  a. In the brains of poly 32 + / + mice (left) on embryonic days 14 and 5, localization of poly 32 gene mRNA is seen in cells around the ventricle, whereas in poly? 2-/-mice (right) They are not found in the brain.
b. 生後 5週齢の poly ?2+/+マウス （左） の脳では、 側脳室の上衣細胞 （矢印） と脈絡叢（矢頭）で poly；52遺伝子 m Aの局在が見られるが、 poly ?2-/-マウス（右） の脳ではそれらは見られない。  b. In the brain of a 5-week-old poly-2 + / + mouse (left), the poly; 52 gene mA is localized in the ependymal cells of the lateral ventricle (arrow) and the choroid plexus (arrowhead). They are not seen in the brain of poly? 2-/-mice (right).
c. 生後 5週齢の poly/? 2+/+マウス （左） の脳では、 中脳水道の上衣細胞で poly ?2遺伝子 mRNAの局在が見られるが、 poly/52- /-マウス（右）の脳では見られない。 *は中脳水道腔を表す。  c. In the brain of a 5 week old poly /? 2 + / + mouse (left), the poly-2 gene mRNA is localized in the ependymal cells of the midbrain aqueduct, but the poly / 52-/-mouse ( Not seen in the right) brain. * Represents midbrain aqueduct.
d. 生後 7週齢の poly ? 2+/+マウス （左） の精巣では poly/? 2遺伝子 mRNAの局在が 見られるが、 poly ?2-/-マウス （右） では見られない。 *は精細管の管腔を表す。 図 7  d. In the testis of 7-week-old poly? 2 + / + mice (left), poly /? 2 gene mRNA is localized, but not in poly? 2-/-mice (right). * Represents the lumen of the seminiferous tubule. Fig. 7
a、 b. 精巣上体尾部から得られた精子を位相差顕微鏡で観察した写真である。 生後 9週齢の poly ?2+/+マウスの精子 (a)の形状に比べ、 poly/? 2-/-マウスの精子 (b)は、 短い尾部をもつもの、 尾部をもたないもの、 円形頭部をもつものなどの奇 形精子が見られた。  a, b. Photographs of a sperm obtained from the epididymis tail observed with a phase contrast microscope. Compared to the shape of spermatozoa of 9-week-old poly? 2 + / + mice (a), the spermatozoa of poly /? 2-/-mice (b) have short tails, those without tails, Malformed spermatozoa such as those with a circular head were seen.
c-h. 精巣の病理解析を行った結果を示した写真である。  c-h are photographs showing the results of pathological analysis of testis.
poly ?2- /-マウス精巣（d)は、 poly/52+Λマウス精巣 (c)と同様の精細管構造を 有する。  The poly? 2-/-mouse testis (d) has the same tubule structure as the poly / 52 + Λ mouse testis (c).
poly/52- /-マウス精巣（f) では、 poly ?2+/+マウス精巣 (e)と同様に、 パキテン 期精母細胞の減数分裂が見られる。  In the poly / 52 − / − mouse testis (f), meiosis of pachytene spermatocytes is observed as in the case of the poly-2 + / + mouse testis (e).
poly ?2+/+マウス精巣 （g) では、 成熟し伸長した核を持ち伸長した尾部を伴う 精子細胞が見られるのに対し、 poly/? 2-/-マウス精巣 （h) では成熟した精子細胞 はわずかに認められる程度であり、未熟な精子細胞がセルトリ細胞から遊離してい る。 In the poly-2 + / + mouse testis (g), sperm cells with a mature and elongated nucleus and an elongated tail are seen, whereas in the poly /? 2-/-mouse testis (h), the mature sperm Cells are only slightly visible and immature sperm cells are released from Sertoli cells. You.
i s j . 精巣上体体部の病理解析を行った結果を示した写真である。 .  3 is a photograph showing the results of pathological analysis of the epididymis body part. .
poly 32+/+マウス（i)では、成熟した精子細胞が見られるのに対し、 poly/? 2-/- マウス （j) では、 成熟した精子細胞は少なく、 未熟な精子細胞とェォジン好性の 物質が多く見られた。  Poly 32 + / + mice (i) show mature sperm cells, whereas poly /? 2-/-mice (j) have few mature sperm cells, immature sperm cells and eosinophils Many substances were found.
図 8は、 poly ?2- /-マウスの生存曲線を示す。  FIG. 8 shows the survival curve of poly 2 − / − mice.
図 9  Fig. 9
a . マウスの poly/52遺伝子を不活性化するために用いた相同組み換え用べクタ —の構築を示す図である。  a. Construction of the homologous recombination vector used to inactivate the mouse poly / 52 gene.
b .相同組み換えベクターを導入した ES細胞クローンにおける変異遺伝子の挿入 をサザン解析により検出した結果を示す写真である。 ' c . poly ?2- /-マウスの精巣における poly^Z遺伝子転写産物（mRNA) の発現を ノ一ザンブロット解析により検出した結果を示す写真である。対照として EF- 1 を 用いた。 .  b. Photographs showing the results of Southern analysis detecting the insertion of the mutant gene in the ES cell clone into which the homologous recombination vector was introduced. FIG. 4 presents photographs showing the results of Northern blot analysis of the expression of poly ^ Z gene transcript (mRNA) in testis of 'c. poly? 2-/-mice. EF-1 was used as a control. .
図 1 0は、 poly ?2- /-マウスと poly ?2+/+マウスの内臓配置を示した解剖写真で ある。 内臓配置をわかりやすくするために肋骨および肝臓を切除してある。 poly β 2-/-マウスの内臓配置は、 poly 2+/+マウスの内臓配置の鏡像対称となっている c 図中、 Apは Apex (尖) 、 Fsは Forestomach (前胃) 、 Spは Spleen (脾臓) を示す記 号である。  FIG. 10 is an anatomical photograph showing the internal organ arrangement of poly-2 − / − and poly-2 + / + mice. Ribs and liver have been resected for better visibility of internal organs. The internal organs of poly β 2 − / − mice are mirror images of the internal organs of poly 2 + / + mice. c In the figure, Ap is Apex (apical), Fs is Forestomach (foregoma), and Sp is Spleen. (Spleen).
図 1 1は、 poly ?2-/-マウスの線毛構造を電子顕微鏡で観察した写真である。上 段は脳室上衣細胞の線毛の断面、 下段は気管上皮細胞の線毛の断面を示している。 poly ?2- /-マウスでは、脳室上衣細胞の線毛（右上）、気管上皮細胞の線毛（右下） において、 ダイニン内腕の欠損が見られる。  FIG. 11 is a photograph of the pilus structure of a poly-2 /-mouse observed with an electron microscope. The upper part shows the cross section of the fimbria of the ependymal cells of the ventricle, and the lower part shows the cross section of the pili of the tracheal epithelial cells. In poly? 2- /-mice, dynein inner arm deficiency is found in the pili of ependymal cells (upper right) and the pili of tracheal epithelial cells (lower right).
図 1 2は、 二連微小管の二本のダイニン腕 （外腕と内腕） の模式図である。  Figure 12 is a schematic diagram of two dynein arms (outer arm and inner arm) of a double microtubule.
図 1 3 ,  Fig. 13,
a . 対照群である poly ?2+/_マウスにおいて、 鼻腔 ·副鼻腔に空洞が認められた ことを示す写真である。 a. Cavity was found in the nasal cavity and paranasal sinuses in the control group, poly? 2 + / _ mice It is a photograph showing that.
b . poly ?2-/-マウスにおいて、 鼻腔 '副鼻腔内に内容物が認められたことを示 す写真である。  b. Photographs showing that the contents were found in the nasal sinuses' sinuses of poly? 2-/-mice.
c poly ?2- /-マウスにおける鼻腔 ·副鼻腔内の拡大写真である。鼻腔内には分 葉好中球が充満し、 粘膜下組織には形質細胞浸潤と毛細血管増生がみられる。 発明を実施するための最良の形態  It is an enlarged photograph of the nasal cavity and the paranasal sinuses in c poly? 2-/-mice. The nasal cavity is filled with lobulated neutrophils, and the submucosa has plasma cell infiltration and capillary hyperplasia. BEST MODE FOR CARRYING OUT THE INVENTION
以下、本発明を実施例により具体的に説明するが、本発明はこれら実施例に制限 されるものではない。  Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.
[実施例 1 ] poly ?2遺伝子の相同組み換えベクターの作製  [Example 1] Construction of homologous recombination vector for poly-2 gene
本実施例では、マウス poly^2遺伝子の相同組み換えべクタ一を構築するために、 まずマウス poly 52ゲノム遺伝子のクロ一ニングを行った。 129SvJマウス肝臓ゲノ ム DNAのライブラリーを用いて、 マウス poly ?2 c DNAをプローブとしてプラークハ ィブリダイゼ一シヨンを行い、 poly/? 2遺伝子ェクソン 1を含む約 20kbのゲノム DNA 断片を単離し、 制限酵素部位のマッビングを行った。  In this example, to construct a homologous recombination vector for the mouse poly ^ 2 gene, first, the mouse poly52 genomic gene was cloned. Using a library of 129SvJ mouse liver genomic DNA, plaque hybridization was performed using mouse poly-2c DNA as a probe, and a genomic DNA fragment of approximately 20kb containing poly / -2 gene exon 1 was isolated. The site was mapped.
相同組み換えにより poly ? 2遺伝子のェクソン 1からェクソン 6をネオマイシン 耐性遺伝子発現カセットと置換するよう、ネオマイシン耐性遺伝子発現カセットの 両端にェクソン 1の 5'側配列を含む遺伝子断片およびェクソン 7、 8を含む遺伝子 断片を結合することで相同領域を配し、さらに 3'下流側に tk発現カセットを結合し、 相同組み換えべクタ一を作成した （図 9 a上段） 。 poly ?2遺伝子座は、 ェクソン 1 の上流に制限酵素 Xbal認識部位 ( X ) があり、 ェクソン 3と 4の間に制限酵素 Spel 認識部位 （Sp) をもつ （図 9 a中段） 。 相同組み換えにより poly ?2遺伝子のェクソ ン 1からェクソン 6がネオマイシン耐性遺伝子発現カセットと置換されると、ェク ソン 1の上流にある Xbal認識部位は保存されるが、ェクソン 3とェクソン 4の間にあ つた Spel認識部位はなくなり、挿入されたネオマイシン耐性遺伝子発現カセット内 に存在する Spel認識部位が生じることになる （図 9 a下段） 。 Xbalおよび Spelで消 化したゲノム DNAに対し、 5' プローブとして表記した部分の塩基配列をプローブと して用いハイブリダイゼ一シヨンを行うと、 正常の poly 52遺伝子座からは 11. Okb 長のバンドが、 変異 poly 52遺伝子座からは 9.2kb長のバンドが検出される。 In order to replace exon 1 to exon 6 of the poly-2 gene with the neomycin resistance gene expression cassette by homologous recombination, include the gene fragment containing the 5 'side sequence of exon 1 and exons 7 and 8 at both ends of the neomycin resistance gene expression cassette The homologous region was arranged by ligating the gene fragments, and the tk expression cassette was further ligated to the 3 'downstream to create a homologous recombination vector (FIG. 9a, upper panel). The poly-2 locus has a restriction enzyme Xbal recognition site (X) upstream of exon 1 and a restriction enzyme Spel recognition site (Sp) between exons 3 and 4 (middle of Fig. 9a). When homologous recombination replaces exon 1 to exon 6 of the poly-2 gene with the neomycin resistance gene expression cassette, the Xbal recognition site upstream of exon 1 is preserved, but between exon 3 and exon 4. The Spel recognition site disappears, and the Spel recognition site present in the inserted neomycin resistance gene expression cassette is generated (Fig. 9a, lower panel). Turn off with Xbal and Spel Hybridization using the nucleotide sequence of the 5 'probe as a probe on the genomic DNA that had been converted, showed that a 11.Okb long band from the normal poly 52 locus A 9.2kb long band is detected from the locus.
[実施例 2 ] 相同組み換えにより変異 poly ?2遺伝子が挿入され poly ?2遺伝子 対の一方が不活性化された ES細胞の樹立 [Example 2] Establishment of ES cells in which a mutated poly-2 gene was inserted by homologous recombination and one of the poly-2 gene pairs was inactivated
本実施例では、相同組み換え用ベクターを、 エレクトロポレーシヨン法によりマ ウス ES細胞 E14 (Hopper, M. et al. : Nature, 326, 292-295, 1987) に導入、 つい で G418、 ガンシクロビルにより選択培養を行った。得られた G418Zガンシクロビル 耐性コロニーについて、 PCRおよびサザンプロヅトにより相同組み換え体の検定を 行い、 poly/? 2遺伝子対の一方に変異 poly 2遺伝子が挿入された細胞株を 4ク口一 ン得た。  In this example, the homologous recombination vector was introduced into mouse ES cell E14 (Hopper, M. et al .: Nature, 326, 292-295, 1987) by electroporation, followed by G418 and ganciclovir. Selection culture was performed. The obtained G418Z ganciclovir-resistant colonies were tested for homologous recombinants by PCR and Southern blotting, and four cell lines in which the mutant poly 2 gene was inserted into one of the poly / -2 gene pairs were obtained.
得られたクローンについてサザン解析により変異遺伝子の挿入の確認を行なつ た （図 9 b) 。 その結果、 変異遺伝子の挿入を行っていない ES細胞株 E14では、 llkb のバンドのみが検出されたのに対し、相同組み換え用ベクターの導入および薬剤選 択により得られた細胞株 96、 97、 110では、 正常 poly ?2遺伝子座に由来する llkb のバンドおよび、 変異 poly/? 2遺伝子座に由来する 9, 2kbのバンドが検出された。  The resulting clones were confirmed by Southern analysis for insertion of the mutant gene (FIG. 9b). As a result, in the ES cell line E14 in which the mutant gene was not inserted, only the llkb band was detected, whereas the cell lines 96, 97, and 110 obtained by introducing the vector for homologous recombination and selecting the drug were used. In, a llkb band derived from the normal poly-2 locus and a 9.2-kb band derived from the mutant poly / -2 locus were detected.
[実施例 3 ]相同組換えにより変異 0^ ?2遺伝子が揷入され 0^ ?2遺伝子対の 双方が不活性化された ES細胞の樹立 [Example 3] Establishment of ES cells in which a 0 ^? 2 gene was mutated by homologous recombination and both 0 ^? 2 gene pairs were inactivated
相同組換え用のベクターの導入により変異 poly/? 2遺伝子が挿入され poly/52遺 伝子の一方が不活性化された ES細胞株を高濃度 G418による選別を行うことにより、 もう一方の遺伝子対も不活性化された細胞株、 即ち本発明のマウス ES細胞株を 2ク ローン得た。  High-concentration G418 selection of ES cell lines in which the mutant poly /? 2 gene has been inserted and one of the poly / 52 genes has been inactivated by the introduction of a vector for homologous recombination, Two clones of a pair of inactivated cell lines, that is, the mouse ES cell line of the present invention were obtained.
[実施例 4 ] poly/? 2遺伝子対の一方が不活性化された ES細胞によるキメラマウ スの作製 [Example 4] Chimeric mouse with ES cells in which one of the poly /? 2 gene pairs was inactivated Production
poly^2遺伝子の一方が不活性化された ES細胞株を、 C57BL/6マウス由来の胚盤胞 にィンジェクシヨンしキメラマウスを得た。  ES cells in which one of the poly ^ 2 genes was inactivated were injected into blastocysts derived from C57BL / 6 mice to obtain chimeric mice.
[実施例 5] poly ?2遺伝子対の双方が不活性化されたマウスの作製 [Example 5] Generation of mouse in which both poly-2 gene pairs were inactivated
poly ?2遺伝子対の一方が不活性化された ES細胞を用いて作製されたキメラマウ スを C57BL/6マウスと交配し、 poly/? 2遺伝子対の一方が不活性化された細胞から成 るマウス （以下、 「poly?2+/-マウス」 と称する） を得た。 これらの poly ?2+/- マウス同士を交配することにより、 poly^2遺伝子対の双方が不活性化されたマウ ス （以下、 「poly ?2 -/-マウス」 と称する） を得た。 これらのマウスの poly ?2 遺伝子対の双方に変異遺伝子が揷入されていることをサザン解析により確認した。  A chimeric mouse prepared using ES cells in which one of the poly-2 gene pairs has been inactivated is crossed with C57BL / 6 mice, and one of the poly-2 genes pairs consists of inactivated cells A mouse (hereinafter, referred to as "poly? 2 +/- mouse") was obtained. By crossing these poly? 2 +/- mice, mice in which both poly ^ 2 gene pairs were inactivated (hereinafter referred to as "poly? 2-/-mice") were obtained. Southern analysis confirmed that the mutant gene was introduced into both poly-2 gene pairs of these mice.
[実施例 6] poly ^2 _/-マウスの特徴 [Example 6] Characteristics of poly ^ 2 _ /-mouse
(1) 逆位  (1) Inversion
poly ?2 -/-マウスにおいて、 23例中 3例で、 心臓および内臓が鏡像対称となる完 全内臓逆位を示す個体が見られた （図 10) 。  In poly? 2-/-mice, 3 out of 23 cases showed individuals showing complete visceral inversion in which the heart and internal organs were mirror-image-symmetric (Fig. 10).
( 2 ) 水頭症  (2) Hydrocephalus
poly ? 2 -/-マウスは水頭症を発症する。水頭症の程度には個体差が見られるが、 重症のものでは生後 2週間目から頭部の膨隆が顕著となり （図 3a、 b, c)、 脳室の 拡張が見られる （図 3c、 4b) 。 また、 外見上頭部の膨隆を示さない個体において も、 組織標本を作成し観察すると、 側脳室の拡張が認められる。  poly? 2-/-mice develop hydrocephalus. Although the degree of hydrocephalus varies from individual to individual, in severe cases, head swelling becomes noticeable from the second week after birth (Fig. 3a, b, c), and ventricular dilatation is observed (Fig. 3c, 4b). ). In addition, even in individuals without apparent head bulge, dilatation of the lateral ventricle is observed when a tissue specimen is prepared and observed.
側脳室の拡張すなわち水頭症の発症は、胎生 10.5日から 18.5日では認められない c しかしながら、外見上は正常であるが生後 1日目には認められるようになる （図 4 a) 。 この時、 大脳皮質は正常であり （図 4a) 、 圧迫はされているが海馬、 視床、 中脳、 小脳、 随脳は保存されている。  Lateral ventricular dilation, or the onset of hydrocephalus, is not observed between embryonic days 10.5 and 18.5. C However, it is apparently normal, but becomes apparent on day 1 of life (FIG. 4a). At this time, the cerebral cortex is normal (Fig. 4a), and the hippocampus, thalamus, midbrain, cerebellum, and cerebrum are preserved, although they are compressed.
生後 5週齢までにさらに側脳室の拡張が進み、大脳皮質の薄層化や脳梁の希薄化 が認められるようになる （図 4b) 。 By 5 weeks of age, the lateral ventricle further expands, thinning of the cerebral cortex and thinning of the corpus callosum (Fig. 4b).
顕著な側脳室拡張を起こした成体マウスでは、脳室周囲の上衣細胞においてアポ トーシスが見られる。このようなアポトーシスは脳室拡張前の脳室周辺においては 認められない。  Adult mice with marked lateral ventricular dilation show apoptosis in ependymal cells around the ventricle. Such apoptosis is not observed around the ventricle before ventricular dilation.
脳室の拡張は、側脳室と第三脳室で認められるが、第四脳室および中脳水道では 認められない。また、胎生 14.5日以降から第三脳室および中脳水道の上衣細胞の過 形成が見られる （図 4 c- e) 。  Ventricular dilatation is seen in the lateral and third ventricles, but not in the fourth and middle ventricles. In addition, hyperplasia of ependymal cells in the third ventricle and the middle cerebral aqueduct is observed from day 14.5 onward (Fig. 4c-e).
poly /?2遺伝子が不活性化されていない正常個体では、 脳室上衣細胞は脳室を取 り卷くように並んだ数層の細胞層を構成しているが （図 5 g、 h) 、 poly ?2 -トマ ウス成体の脳では第三脳室および中脳水道の上衣細胞の過形成が見られ層構造が 破綻している部分が認められる （図 5 a-f) 。 これら上衣細胞過形成部位では、 球 形で明確な核を持つ単核の細胞とェォジン好性の細胞質を持つ単核の細胞が認め. られる （図 5 c) 。  In normal individuals in which the poly /? 2 gene is not inactivated, the ventricular ependymal cells comprise several layers of cells that surround the ventricle (Fig. 5g, h). On the other hand, in the adult poly-2-Thomas mouse brain, hyperplasia of the ependymal cells of the third ventricle and the middle cerebral aqueduct was observed, and the layer structure was disrupted (Fig. 5 af). At these ependymal hyperplasia sites, mononuclear cells with a spherical and well-defined nucleus and mononuclear cells with eosinphilic cytoplasm are observed (Fig. 5c).
水頭症を示している脳室の上衣細胞を電子顕微鏡で観察すると、上衣細胞の多層 化および微絨毛縁の崩壊が見られる （図 4 f) 。  Electron microscopy of ependymal cells showing hydrocephalus reveals multilayering of ependymal cells and disruption of the microvilli margin (Fig. 4f).
水頭症の原因の一つとして脳脊髄液の循環経路における閉塞が考えられるため、 脳室内にトリパンブル一を注入し脊柱管への流入を調べる手法 （Genes &  One of the causes of hydrocephalus is obstruction in the cerebrospinal fluid circulation path. Therefore, a technique of injecting trypan blue into the ventricle and examining the influx into the spinal canal (Genes &
Development, 12, 1092-1098, 2000) を用いて閉塞の有無を調べたところ、 脳脊髄 液の排液経路が開口していないことが判明した。 Development, 12, 1092-1098, 2000), it was found that there was no obstruction in the drainage pathway of cerebrospinal fluid.
双方の poly ?2遺伝子が不活性化されたマウスにおける水頭症の発症原因は、 脳 脊髄液循環経路の閉塞および上衣細胞の微絨毛縁の崩壊による、脳脊髄液の産生お よびあるいは再吸収における異常であると考えられる。  The onset of hydrocephalus in mice in which both poly-2 genes are inactivated is due to cerebrospinal fluid production and / or reabsorption due to occlusion of the cerebrospinal fluid circulation and disruption of ependymal microvillous margins. It is considered abnormal.
( 3 ) 発育不良  (3) poor growth
水頭症を発症した poly^2 - /-マウスは、 poly^2遺伝子が不活性化されていない 野生型マウス （以下「poly 52+/+マウス」 と称する） に比べて発育遅延を示す （図 3 a) 。 生後 1 4日目、 2 1日目において、 poly ? 2-/-マウスの体重は poly 2+/+ マウスの 60〜80%程度である。 Poly ^ 2-/-mice with hydrocephalus show growth retardation compared to wild-type mice in which the poly ^ 2 gene is not inactivated (hereinafter referred to as "poly52 + / + mice"). 3 a). On days 14 and 21 after birth, poly? 2-/-mice weighed poly 2 + / + About 60-80% of mice.
( 4 ) 生存曲線 '  (4) Survival curve ''
離乳後の観察によると、 poly ^2-/-マウスは、 生後 3週齢から死亡し始め、 5週 齢までには生存率が 50%となる （図 8) 。 死亡する poly ?2-/-マウスは大部分が水 頭症を示す個体であり、 5週齢を越えて生存する poly 52- /-マウスは、外見上は正 常マウスと同一である。  According to postweaning observations, poly ^ 2-/-mice begin to die at 3 weeks of age and have a 50% survival rate by 5 weeks of age (Figure 8). The dead poly 2 − / − mice are mostly individuals with hydrocephalus, and the poly 52 − / − mice surviving beyond 5 weeks of age are apparently identical to normal mice.
( 5 ) 脳における poly 52遺伝子の発現  (5) Expression of poly52 gene in brain
poly 52遺伝子の転写産物 （mRNA) の局在を in situ,、ィブリダイゼーシヨンによ り調べた。 poly ?2mRNAは、 野生型マウスの胎児脳の脳室周辺の細胞および成体の 脳室上衣細胞および脈絡叢に局在しており、 poly 52-/-マウスではそれらの細胞に おける poly ?2mRNAの局在は認められない （図 6a- c) 。  The localization of the transcript (mRNA) of the poly52 gene was examined by in situ and hybridization. poly-2 mRNA is localized in cells around the ventricle of the fetal brain of wild-type mice and in adult ependymal ependymal cells and choroid plexus.In poly52-/-mice, poly-2 mRNA is expressed in those cells. No localization is observed (Figures 6a-c).
( 6 ) 精巣における poly^2遺伝子の発現  (6) Expression of poly ^ 2 gene in testis
poly/? 2遺伝子の転写産物 （mRNA) の局在を in situハイブリダイゼーシヨンによ り調べた。 poly ?2mMAは、野生型マウスの精巣での局在が認められる。 poly/52-/- マウスでは精巣における poly ?2mRNAの局在は認められない （図 6d) 。 .  The localization of the transcript (mRNA) of the poly /? 2 gene was examined by in situ hybridization. Poly-2mMA is localized in the testes of wild-type mice. In poly / 52-/-mice, no localization of poly-2 mRNA in testis was observed (Fig. 6d). .
また、 ノーザンプロヅト解析により poly ?2- /-マウスの精巣における poly ?2遺 伝子転写産物（mRNA)の発現解析を行なった（図 9 c)。 poly ?2+/+マウスおよび、 (poly ?2+/-マウスでは、 poly/? 2遺伝子 mRNAの発現が認められるが、 poly/? 2 -/- マウスでは、 poly ?2遺伝子の発現は認められない。図 9 c下段は EF-1ひの発現を示 したものであり、これにより各レーンに RNAサンプルが存在することが証明される。  The expression of poly-2 transcripts (mRNA) in testis of poly-2 /-mice was analyzed by Northern blot analysis (Fig. 9c). In poly? 2 + / + mice and (poly? 2 +/- mice, the expression of poly /? 2 gene mRNA is observed, but in poly /? 2-/-mice, the expression of poly? 2 gene is observed. The lower panel in Fig. 9c shows the expression of EF-1 cells, which demonstrates the presence of the RNA sample in each lane.
( 7 ) 生殖能力  (7) Fertility
poly ?2-/-マウスの生殖能力について調べた。 6例の poly ?2- /-マウスそれそれ を 2例ずつの C57BL/6Jメスマウスと同居させ、 30日間妊娠の成立の有無を観察した。 その結果、いずれも交配成立を示す膣栓の形成は観察されるものの産仔は得ら な かった。一方、 poly ?2- /-メスマウスは、 C57BL/6Jォスマウスあるいは poly ?2+/- ォスマウスとの交配が成立し、 3例の poly ?2-/-メスマウスで産仔が得られた。 こ れらのこのことから poly ? 2-/-ォスマゥスは不妊であるが、 poly ? 2-/-メスマウス は正常の生殖能力をもっといえる。 Poly? 2-/-mice were examined for fertility. Six poly-2 /-mice were co-resident with two C57BL / 6J female mice, and the presence or absence of pregnancy was observed for 30 days. As a result, the formation of vaginal plugs indicating the establishment of mating was observed, but no offspring were obtained. On the other hand, poly-2 /-female mice crossed with C57BL / 6J os mice or poly-2 +/- os mice, and offspring were obtained from 3 poly-2 /-female mice. This From these facts, poly 2-/-mice are infertile, whereas poly 2-/-female mice have more normal fertility.
( 8 ) 精子および精巣の解析  (8) Analysis of sperm and testis
poly ? 2-/-ォスマゥスは不妊であったことから、精子および精巣の解析を行った c po ly ^ 2- /-ォスマウスの精巣上体尾部から得た精子は運動能力を欠損しており、頭 部のみ、奇形頭部を有する、尾部が短いといった奇形精子が多く見られた（図 7 b)。 精巣の病理解析を行ったところ、 poly/? 2-/-の精巣は、 12段階に分けられる精子形 成過程すベての段階を有する精細管を有し （図 7 d) 、 また、 パキテン期精母細胞 の減数分裂が見られ （図 7 f ) 、 これらは poly ?2+/+のそれ （図 7 c、 e) と同様で あった。 しかしながら、 poly ?2-/-の精巣では未熟な精子細胞がセルトリ細胞から 遊離しており、成熟し伸長した核をもつ精子細胞はわずかに認められる程度であつ た （図 7 h) 。 poly 52- /-の精巣上体体部中には、 成熟した精子細胞数は少なく、 未熟な精子細胞とェォジン好性の物質が多く見られた （図 7 j) 。  Since poly? 2-/-osma was infertile, spermatozoa and testis were analyzed.The sperm obtained from the epididymis of the c po ly ^ 2- /-os mice lacked motor ability. Many malformed spermatozoa with a malformed head and a short tail were found only in the head (Fig. 7b). Pathological analysis of the testes revealed that the testes of poly /? 2-/-had seminiferous tubules with all stages of spermatogenesis divided into 12 stages (Fig. 7d). Meiosis of spermatocytes was observed (Fig. 7f), which was similar to that of poly-2 + / + (Fig. 7c, e). However, immature sperm cells were released from Sertoli cells in the poly-2 /-testis, and only a few sperm cells with mature and elongated nuclei were observed (Fig. 7h). In the epididymis of poly52-/-, the number of mature spermatids was low, and immature spermatids and eosin-friendly substances were many (Fig. 7j).
poly ?2-/-マウス精子の核の成熟度を調べるため、未受精卵の細胞質内へ精子頭 部を顕微注入することによって受精させ（顕微受精）、後の胚発生を調べた。 B6C3F1 系統の未受精卵に poly ?'2- /-マウスの精子頭部を顕微注入し、 受精の成立した 23 個の受精卵を偽妊娠メスの卵管内に移植し、 妊娠を継続させた。 その結果、 12匹の 産仔が得られこれらは正常に成長した。 このことから、 poly^2- /-マウス精子の核 成熟は正常に起こっていると考えられる。  To examine the maturity of the nuclei of poly? 2-/-mouse sperm, unfertilized eggs were fertilized by microinjection of the sperm head into the cytoplasm (microinsemination), and subsequent embryo development was examined. Micro-injection of the sperm head of a poly? '2-/-mouse into unfertilized eggs of the B6C3F1 strain was performed, and 23 fertilized eggs that had been fertilized were transplanted into the oviduct of a pseudopregnant female to continue pregnancy. The result was 12 offspring, which grew normally. This suggests that poly ^ 2-/-mouse sperm nucleation is normal.
( 9 ) 電子顕微鏡による線毛構造の解析  (9) Analysis of pili structure by electron microscope
poly ?2-/-マウスにおいて、 内臓逆位、 水頭症、 精子の運動不全が見られ、 線毛 運動の異常が疑われたため、 電子顕微鏡による線毛構造の解析を行った。 poly^ 2+/+マウスの脳室上衣細胞（図 1 1左上） および気管上皮細胞（図 1 1左下） の線 毛の断面で見られるように、 正常な線毛は、 一組の単微小管対と 9組の周辺ニ連微 小管から り、 二連微小管は二本のダイニン腕 （外腕と内腕） を持つ （図 1 2 ) 。 poly^2- /-マウスの脳室上衣細胞（図 1 1右上）および気管上皮細胞（図 1 1右下） の線毛の断面では、 ダイニン内腕が欠損している様子が観察された。 In poly? 2-/-mice, visceral inversion, hydrocephalus, and sperm dysmotility were observed, suggesting abnormal ciliary motility. The pilus structure was analyzed by electron microscopy. As seen in the cross-sections of the pilus of polyventricular ependymal cells (Fig. 11, upper left) and tracheal epithelial cells (Fig. 11, lower left) in poly ^ 2 + / + mice, normal pili consist of a set of The double microtubule has two dynein arms (outer arm and inner arm), consisting of a tube pair and nine sets of peripheral double microtubules (Fig. 12). Ventricular ependymal cells (Fig. 11, upper right) and tracheal epithelial cells (Fig. 11, lower right) of poly ^ 2-/-mice In the cross-section of the fimbriae, it was observed that the inner arm of dynein was missing.
( 1 0 ) 慢性副鼻腔炎  (10) Chronic sinusitis
poly β 2_/-マウスにおいてダイニン内腕の欠損が見られたことから、 Kartagener 症候群の症状のひとつである慢性副鼻腔炎の検索を poly ?2-/-マウスにて行った。 対照群である poly ?2+/-マウスでは鼻腔'副鼻腔に空洞が認められたが（図 1 3 a )、 poly ^2-/-マウスでは鼻腔 ·副鼻腔内に内容物が認められた （図 1 3 b ) 。拡大す ると、 鼻腔内には分葉好中球が充満し、粘膜下組織には形質細胞浸潤と毛細血管増 生がみられ（図 1 3 c )、 poly ?2-/-マウスは慢性化膿性鼻腔炎を発症していると 診断された。気管上皮細胞や脳室上衣細胞の繊毛で見られたのと同様の繊毛構造異 常（ダイニン内腕欠損） が、 鼻腔粘膜を構成する鼻粘膜繊毛細胞の繊毛でも起こつ ているために運動障害が起こり、 鼻腔炎を発症しているものと推察される。 産業上の利用の可能性  Since poly-β 2 _ /-mice lacked the inner arm of dynein, chronic sinusitis, a symptom of Kartagener syndrome, was searched for in poly-2 /-mice. In the control group poly-2 +/- mice, cavities were found in the nasal sinuses' sinuses (Fig. 13a), whereas in poly ^ 2-/-mice, the contents were found in the nasal cavities and sinuses. (Figure 13b). When expanded, the nasal cavity was filled with lobulated neutrophils, the submucosa showed plasma cell infiltration and hyperplasia (Fig. 13c), and the poly? 2-/-mice were chronic. She was diagnosed with purulent rhinitis. Dyskinesia due to ciliary structural abnormalities (dynein inner arm defect) similar to those found in cilia of tracheal epithelial cells and ventricular ependymal cells also occur in the cilia of nasal mucociliary cells that make up the nasal mucosa It is presumed that rhinitis has developed. Industrial applicability
本発明により、 poly ?2遺伝子の発現が人為的に抑制された ES細胞およびマウス が提供され、 該マウスが、 水頭症、 発育不良、 内臓逆位、 精子の成熟不良、 脳室上 衣細胞および気管上皮細胞の線毛における繊毛構造異常（ダイニン内腕欠損） を特 徴とすることが明らかとなった。従って、 本発明の遺伝子改変 ES細胞、 遺伝子改変 動物、 あるいは該動物から樹立した細胞株を利用すれば、 poly/? 2遺伝子の機能の 解析の他、 水頭症、 発育不良、 内臓逆位、 精子の成熟不良、 i腿 otile cilia症候群 などの疾患の解明やこれら疾患の治療薬などのスクリーニングを行なうことも可 能である。 また、 poly ?2遺伝子及びタンパク質は上記疾患の治療薬などとして利 用することが可能であると考えられる。  According to the present invention, there are provided ES cells and mice in which poly-2 gene expression is artificially suppressed, wherein the mice have hydrocephalus, poor growth, visceral inversion, poor sperm maturation, ventricular ependymal cells and It was found to be characterized by abnormal ciliary structure (dynein inner arm defect) in the pili of tracheal epithelial cells. Therefore, if the genetically modified ES cells, genetically modified animals, or cell lines established from the animals of the present invention are used, the function of the poly /? 2 gene can be analyzed, as well as hydrocephalus, poor growth, visceral inversion, and sperm. It is also possible to elucidate diseases such as poor maturation and itile otile cilia syndrome and to screen for remedies for these diseases. In addition, it is considered that poly-2 gene and protein can be used as therapeutic agents for the above-mentioned diseases and the like.

Claims

請求の範囲 poly ?2遺伝子の発現が人為的に抑制されていることを特徴とするげつ歯類 ES細胞。  Claims A rodent ES cell, wherein the expression of the poly-2 gene is artificially suppressed.
poly ?2遺伝子の遺伝子対の一方または双方に外来遺伝子が挿入されている ことを特徴とするげつ歯類 ES細胞。  A rodent ES cell comprising a foreign gene inserted into one or both of the poly-2 gene pair.
げっ歯類 ES細胞がマウス ES細胞である請求項 1または 2記載の ES細胞。 poly ?2遺伝子の発現が人為的に抑制されていることを特徴とする遺伝子改 変げつ歯類動物。  3. The ES cell according to claim 1, wherein the rodent ES cell is a mouse ES cell. Genetically modified rodents characterized in that expression of the poly-2 gene is artificially suppressed.
poly ?2遺伝子の遺伝子対の一方または双方に外来遺伝子が挿入されている ことを特徴とするげっ歯類動物。  A rodent animal characterized in that a foreign gene has been inserted into one or both of the poly-2 gene pairs.
げっ歯類動物がマウスである請求項 4または 5記載の動物。  6. The animal according to claim 4, wherein the rodent is a mouse.
請求項 4〜 6のいずれかに記載の動物から樹立された細胞株。  A cell line established from the animal according to any one of claims 4 to 6.
PCT/JP2001/009204 2000-10-20 2001-10-19 POLYβ2 KNOCKOUT ANIMALS WO2002033054A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2002536424A JPWO2002033054A1 (en) 2000-10-20 2001-10-19 polyβ2 gene deficient animal
AU2001295982A AU2001295982A1 (en) 2000-10-20 2001-10-19 Polybeta2 knockout animals

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP2000-321714 2000-10-20
JP2000321714 2000-10-20
JP2000-372555 2000-12-07
JP2000372555 2000-12-07
JP2001-120294 2001-04-18
JP2001120294 2001-04-18

Publications (1)

Publication Number Publication Date
WO2002033054A1 true WO2002033054A1 (en) 2002-04-25

Family

ID=27345002

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2001/009204 WO2002033054A1 (en) 2000-10-20 2001-10-19 POLYβ2 KNOCKOUT ANIMALS

Country Status (3)

Country Link
JP (1) JPWO2002033054A1 (en)
AU (1) AU2001295982A1 (en)
WO (1) WO2002033054A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005017155A1 (en) 2003-06-18 2005-02-24 Chugai Seiyaku Kabushiki Kaisha Fucose transporter
EP2208783A1 (en) 2004-12-22 2010-07-21 Chugai Seiyaku Kabushiki Kaisha Method of producing an antibody using a cell in which the function of fucose transporter is inhibited
US7829757B2 (en) 2001-10-24 2010-11-09 Chugai Seiyaku Kabushiki Kaisha SGRF gene-modified mouse

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025442A1 (en) * 1999-10-01 2001-04-12 Consejo Superior De Investigaciones Cientificas Dna polymerase lambda and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001025442A1 (en) * 1999-10-01 2001-04-12 Consejo Superior De Investigaciones Cientificas Dna polymerase lambda and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AOUFOUCHI S. ET AL.: "Two novel human and mouse DNA polymerases of the polX family", NUCLEIC ACIDS RES., vol. 28, no. 18, 15 September 2000 (2000-09-15), pages 3684 - 3693, XP002157837 *
LINDEMAN G.J. ET AL.: "A specific, nonproliferative role for E2F-5 in choroid plexus function revealed by gene targeting", GENES DEV., vol. 12, no. 8, 1998, pages 1092 - 1098, XP002907709 *
NAGASAWA K. ET AL.: "Identification and characterization of human DNA polymerase beta2, a DNA polymerase beta-related enzyme", J. BIOL. CHEM., vol. 275, no. 40, 6 October 2000 (2000-10-06), pages 31233 - 31238, XP001011150 *
TAKAI H. ET AL.: "Aberrant cell cycle checkpoint function and early embryonic death in Chk1(-/-) mice", GENES DEV., vol. 14, no. 12, 15 June 2000 (2000-06-15), pages 1439 - 1447, XP002907710 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7829757B2 (en) 2001-10-24 2010-11-09 Chugai Seiyaku Kabushiki Kaisha SGRF gene-modified mouse
WO2005017155A1 (en) 2003-06-18 2005-02-24 Chugai Seiyaku Kabushiki Kaisha Fucose transporter
EP2228445A1 (en) 2003-06-18 2010-09-15 Chugai Seiyaku Kabushiki Kaisha Fucose Transporter
EP2208783A1 (en) 2004-12-22 2010-07-21 Chugai Seiyaku Kabushiki Kaisha Method of producing an antibody using a cell in which the function of fucose transporter is inhibited
EP2216404A1 (en) 2004-12-22 2010-08-11 Chugai Seiyaku Kabushiki Kaisha Method of producing an antibody using a cell in which the function of fucose transporter is inhibited

Also Published As

Publication number Publication date
JPWO2002033054A1 (en) 2004-02-26
AU2001295982A1 (en) 2002-04-29

Similar Documents

Publication Publication Date Title
Chen et al. Mutation of the mouse hepatocyte nuclear factor/forkhead homologue 4 gene results in an absence of cilia and random left-right asymmetry.
Brody et al. Ciliogenesis and left–right axis defects in forkhead factor HFH-4–null mice
McMahon et al. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain
Cho et al. The Na+-Ca2+ exchanger is essential for embryonic heart development in mice
CN107164406B (en) Construction method and application of mouse model for conditional knockout of Tmem30a gene in pancreatic islet β cells
Warren et al. A Sall4 mutant mouse model useful for studying the role of Sall4 in early embryonic development and organogenesis
DK2141985T3 (en) Transgenetic pig model of cystic fibrosis
Appelbe et al. Disruption of the mouse Jhy gene causes abnormal ciliary microtubule patterning and juvenile hydrocephalus
US8952214B2 (en) Animal model for chronic obstructive pulmonary disease and cystic fibrosis
US6452065B2 (en) Transgenic mouse expressing non-native wild-type and familial Alzheimer's Disease mutant presenilin 1 protein on native presenilin 1 null background
WO2002033054A1 (en) POLYβ2 KNOCKOUT ANIMALS
KR101954726B1 (en) Animal model for pendred syndrome and method for producing the same
US6225525B1 (en) ATP-binding cassette transporter (ABC1) modified transgenic mice
EP1910416B1 (en) Non-human animal model for cardiovascular disease characterized by a disrupted fibulin-4 gene
CN117230077B (en) Application of Hakai gene in RP disease model construction and construction method
JP4303907B2 (en) Radixin knockout animal
KR100430199B1 (en) Transgenic mouse with disrupted calcium ion channel alpha1D gene and production method thereof
CN115777626A (en) Construction method and application of Ift140 gene rod-like cell conditional knockout mouse model
JP4696316B2 (en) CD9 / CD81 double deficient non-human animal
JP2009225697A (en) Abca12 GENE FUNCTION-DEFICIENT MOUSE
WO2018012497A1 (en) Disease model animal and disease therapeutic agent
JP2008131940A (en) Transgenic nonhuman mammal having mutation in gne gene
JP2007312641A (en) Nonhuman mammal, method for preparing the same and method for screening therapeutic agent for skin disease by using the same
Cho et al. The Na [sup+]-Ca [sup 2+] Exchanger Is Essential for Embryonic Heart Development in Mice.
JP2004357652A (en) Pathological model animal for cataract

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PH PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref country code: JP

Ref document number: 2002 536424

Kind code of ref document: A

Format of ref document f/p: F

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase