WO2006022361A1 - ANIMAL DÉFICIENT EN GÈNE Psf1 ET PROCÉDÉ D'UTILISATION DE CELUI-CI - Google Patents

ANIMAL DÉFICIENT EN GÈNE Psf1 ET PROCÉDÉ D'UTILISATION DE CELUI-CI Download PDF

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WO2006022361A1
WO2006022361A1 PCT/JP2005/015496 JP2005015496W WO2006022361A1 WO 2006022361 A1 WO2006022361 A1 WO 2006022361A1 JP 2005015496 W JP2005015496 W JP 2005015496W WO 2006022361 A1 WO2006022361 A1 WO 2006022361A1
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gene
psfl
deficient
human mammal
animal
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PCT/JP2005/015496
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English (en)
Japanese (ja)
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Nobuyuki Takakura
Masaya Ueno
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National University Corporation Kanazawa University
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Publication of WO2006022361A1 publication Critical patent/WO2006022361A1/fr

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    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases

Definitions

  • the present invention relates to a non-human mammal deficient in the function of the Psfl gene and a method for using the same. Background technology
  • the initiation of DNA replication in eukaryotic cells is controlled by the action of various proteins at the origin of replication.
  • 6 types of proteins (Orel-6) are bound to the replication origin, but Cdc6 and Mem complex bind here to form a pre-replication complex, and further, phosphorylation of Cdc6
  • the Cdc7 / Dbi4 complex then binds to this pre-replication complex, and the subsequent binding of Cdc45 serves as a signal, mobilizing DNA polymerase and starting DNA replication.
  • Dpbl l is a protein involved in the replication initiation mechanism (especially the S phase checkpoint) as a cofactor of DNA polymerase ⁇ .
  • Sld l / Dpb3, Sld2, Sld3, Sld4 / Cdc45, Sld5, Sld6 / Rad53, etc. have been identified as factors that interact with Dpbl l. It has been reported that binding to l is essential for DNA polymerase mobilization (Kamimura, Y., et al., EMBO J., 2001, 20 (8), p2097-2107) o
  • Psi l is a gene found in Dpbl l as a factor that binds to Sld5 (Partner of Sid Five).
  • Araki et al. Reported that a complex consisting of Sld5, Psf L Psf2, and Psf3 is incapable of DNA replication in Saccharomyces cerevisiae (Kubota, Y. et al., Genes & Development 2003, 17, pl l41-1152, Takayama, Y. et al., Genes & Development 2003, 17, pl l53-1165).
  • An object of the present invention is to elucidate the function of Psfl at the individual level, to elucidate various pathological conditions related to Psfl, and to provide a new research tool using Psfl.
  • the inventors established Psfl gene-deficient mice for the purpose of elucidating the functions of Psfl at the individual level. As a result of analyzing this mouse, it was found that the Psfl gene was lethal when it was deficient in homo, but when it was deficient in hetero, serious myelosuppression similar to myelosuppressive disease was observed. Completed the invention.
  • the present invention relates to a non-human mammal deficient in the function of the Psfl gene on the chromosome (Psfl gene deficient animal).
  • the function of the Psfl gene can be deficient by deletion or substitution of at least a part of the sequence on the Psfl gene or its expression control region, and insertion of Z or other sequences.
  • the Psil gene-deficient animal of the present invention is produced, for example, by the following steps.
  • the recombinant ES cell is introduced into an early embryo and developed to obtain a chimeric animal
  • the F1 heterozygous animals obtained by mating the above chimeric animals with wild-type animals are mated with each other, and an animal lacking the function of the Psil gene on the chromosome is obtained from the obtained F2 animals or their progeny.
  • a mouse is particularly preferable.
  • the Psil gene-deficient animal of the present invention can be suitably used not only for functional studies of the Psfl gene but also as a model animal for myelosuppressive disease.
  • myelosuppressive diseases include aplastic anemia, administration of anticancer agents, prolonged myelosuppression due to radiation exposure (irradiation), myelosuppression associated with autoimmune diseases, Examples include bone marrow suppression caused by bacteria and virus infection, bone marrow suppression as a side effect caused by pharmaceuticals, and bone marrow suppression of unknown cause.
  • hematopoietic stem cells can be easily obtained.
  • the obtained hematopoietic stem cells are useful for various research and screening systems.
  • the present invention also provides a screening method for a therapeutic agent for myelosuppressive disease using the Psfl gene-deficient animal of the present invention.
  • the method can evaluate the effect of the test substance as a therapeutic agent for myelosuppressive disease using as an index the difference in expression of the animal between the test substance administration condition and the non-administration condition.
  • an animal deficient in the function of the Psf l gene is provided.
  • This Psf l gene-deficient animal exhibits various symptoms due to bone marrow function suppression, and particularly similar to severe myelosuppression after administration of an anticancer drug by administration of 5-FU or the like. Therefore, it is useful for elucidating the pathology of such diseases, drug development, and drug screening.
  • hematopoietic stem cells can be easily prepared using the Psfl gene-deficient animal of the present invention.
  • Figure 1 shows the primary structure of the Psil protein. From the N-terminal side, there are coiled-coil domain, arginine-rich basic domain, and PEST-like domain.
  • Fig. 2 shows the results of expression analysis of the Psfl gene in mouse tissues and bone marrow cells.
  • BM bone marrow.
  • 3A and 3B show the localization in various tissues of Psfl gene-deficient mice. msn; mesenchymal cells, Hr; heart, AER; apical ectodermal ridge, DA; dorsal aorta.
  • Figure 4 shows the subcellular localization in NIH3T3 cells.
  • Figure 5 shows the construction of the Psil gene deficient evening vector.
  • E EcoRI cleavage site
  • B BamHI cleavage site.
  • DT Diphtheria toxin fragment A gene
  • neo Neomycin resistance gene.
  • Fig. 6A shows the results of Southern plot analysis (upper) and PCR analysis (lower) (upper middle left: wild-type allele, right: mutant allele).
  • Fig. 6B shows HE-stained tissue sections of post-implantation embryos (embryo 6.5 days).
  • Figure 7 shows the results of in vitro culture of Ps gene homo-deficient (-/-) embryos and hetero-deficient (+/-) embryos.
  • Fig. 8 shows the results of culturing embryos for 4 days, incorporating BrdU for 12 hours, and then detecting the incorporated BrdU with a specific antibody after fixation, as in Fig. 7.
  • An ICM that is actively split and incorporated BrdU is indicated by an arrow.
  • Fig. 9 is a graph comparing 9 A: survival rate, 98: blood 1 ⁇ 1] ⁇ (, 9 C: bone marrow MNC after administration of 5-FU in heterozygous mice lacking Psfl gene and wild type mice
  • Figure 10 shows FACS analysis of bone marrow cells after 5-FU administration in Psil gene heterozygous and wild-type mice 1.
  • OA analysis of bone marrow cell morphology by FSC and SSC 10 B is the analysis of the expression of Mac-1 in the stem cell population, and the stem cells are contained in the area enclosed by the square in the figure 10 C is the analysis of the stem cell DNA content
  • This specification includes the contents described in the specification of Japanese Patent Application No. 2004-242022, which is the basis of the priority of the present application.
  • the present invention relates to a non-human mammal deficient in the function of the Psfl gene on the chromosome.
  • the “Psil gene” according to the present invention is a gene found as a factor (Partner of Sid Five) that binds to SLD5 in research on the budding yeast Dpbll.
  • the complex formed by Sld5, Psfl, Psi2, and Psf3 has been reported to be essential for DNA replication in budding yeast, but its detailed function is unknown.
  • mouse Psfl gene has been registered as GenBank Accession No. AK013116 (ypote tical protein KIAA0186 omologue). Was completely consistent with the CDS of mouse Ps isolated and identified by the inventors.
  • the genomic gene of mouse Psil contains 7 exons and 6 introns, and encodes a Psil protein consisting of 196 aa amino acids in total length (SEQ ID NO: 1 is the Psil CDS identified by the present inventor (SEQ ID NO: 2 is the amino acid). Array)).
  • the Psfl gene can be cloned from the homology with a known Psil gene and sequenced by a conventional method. That is, the genome of the animal]) NA library is prepared, and the library is screened using a known Psfl gene derived from the genetically closest species or a part thereof as a probe. Identify the gene and determine its sequence.
  • the “Psil gene” includes all orthologs of the Psil gene as described above, and includes not only genomic DNA but also mRNA and cDNA.
  • “the function of the Psil gene is deficient” means that the Psil gene on the chromosome is destroyed and the function is not normally expressed. That is, not only when the Psfl gene product is not expressed at all, but also when the gene product is expressed, if it does not have a normal function as Psil, it means “the function of the Psil gene is deficient”.
  • Such disruption of the Psfl gene must be caused by alterations such as deletion, substitution, and / or insertion of other sequences on the Psil gene or on its expression control region including its transcriptional regulatory region and promoter region. You can.
  • the site where the deletion, substitution or insertion is performed, and the sequence where the deletion, substitution or insertion is performed are not particularly limited as long as the normal function of the Psil gene can be deleted.
  • the non-human mammal of the present invention has a defect in the Psil gene function in one allele (hetero) on the chromosome. This is because the loss of Psfl gene function in both alleles (homo) is lethal to animals.
  • non-human mammal is a mammal other than human.
  • rodents such as mice, rats, and rabbits are preferred.
  • mice Most preferred are mice in which ES cells are established and genetic recombination can be performed easily.
  • the Psfl gene-deficient animal of the present invention can be produced by using a technique such as gene targeting, Cre-XoX system, or somatic cell clone.
  • Gene targeting is a technique for introducing mutations into specific genes on chromosomes using homologous recombination (Capeccc i, MR Science, 244, 1288-1292, 1989, Thomas, KR & Cpeccchi, MR Cel, 44 419-428, 1986).
  • an evening-getting vector for deleting the Psi l gene is constructed.
  • This genomic DNA library must be an ES cell of the animal used or a library made from genomic DNA of the strain from which the cell is derived so that the frequency of recombination is not reduced due to polymorphisms, etc. .
  • a commercially available library (such as Stratagene's 129Sv / J Genome Library) can be used.
  • the Genome Library is screened using the target Psi cDNA or a partial sequence as a probe. To clone Psi l genomic DNA.
  • the cloned genomic DNA is subjected to sequencing, Southern plotting, restriction enzyme digestion, etc. to create a restriction enzyme map that clearly shows the position of each exoon, and to determine the site of mutation introduction.
  • a probe for screening homologous recombinants is set outside the homologous region used for the targeting vector.
  • the mutation (deletion, substitution, or insertion) introduced on the chromosome is not particularly limited as long as the normal function of the Psi l gene is impaired.
  • the sequence to be deleted or replaced may be an intron region or an exon region of the Psil gene, or an expression control region of the Psil gene.
  • mutations that delete or replace a substantial part of the exon region of the Psf gene can be deleted.
  • other sequences to be inserted are not particularly limited, but the following marker gene sequences are preferably used.
  • the targeting vector contains an appropriate selectable marker for selection of the recombinant along with the 3 'and 5' homologous regions of the mutagenesis site.
  • markers include neomycin resistance genes (pGI (neo, pMC lneo, etc.), positive selection markers such as hygromycin B phosphotransferase gene, LacZ, and expression of genes to be disrupted such as;
  • Negative selection markers such as reporter, simple herpesvirus thymidine kinase gene (HSV-TK), diphtheria toxin ⁇ fragment (DT-A), etc. are not limited to these. Appropriate restriction sites for linearization of the vector are included outside the homologous region.
  • Fig. 5 shows an example of a targeting vector for deletion of the mouse Psi l gene.
  • This construct does not mutate the intron 4 and intron 5 gene sequences of the Psf l gene, and is almost identical to the exon 5 of the Psf l gene (which corresponds to about 20% of the coding region of the mouse Psi l gene). All sequences are constructed to replace the LacZ gene as a reporter gene and the neomycin resistance gene as a positive selection marker.
  • the diphtheria toxin A fragment (DT) is inserted as a negative selection marker.
  • Such a targeting vector can be suitably constructed using a commercially available plasmid vector (for example, pBluescript 11 (manufactured by St ratagene)). 2) Introduction of targeting vector into ES cells
  • the constructed targeting vector is introduced into a totipotent cell such as an embryonic stem cell (ES cell).
  • ES cells have been established in mice, hamsters, pigs, etc.
  • Multiple cell lines, such as strains, are available.
  • embryonic carcinoma cells (EC cells) can be used instead of ES cells.
  • ES cells should be cultured in an appropriate medium prior to the introduction of the evening vector.
  • mouse fibroblasts and the like are used as feeder cells, and a liquid culture medium for ES cells (for example, manufactured by GIBC0) is added and co-cultured. To do.
  • ES cells into which a targeting vector has been introduced can be easily selected with a marker inserted into the vector.
  • a marker inserted into the vector For example, in the case of a cell in which a neomycin resistance gene is introduced as a marker, primary selection can be performed by culturing in a medium for ES cells supplemented with G418.
  • ES cells into which an evening-targeting vector has been introduced a part of the PsiI gene on the chromosome is replaced with the vector by homologous recombination, and the endogenous Psf1 gene is destroyed.
  • Whether or not the desired homologous recombination has been achieved can be determined by dienotype analysis using Southern plotting, PCR method or the like. Southern blotting can be performed using a probe (external probe) set outside the site of mutagenesis. Dienotype analysis by PCR can be performed by detecting specific amplification products of wild-type and mutant Psfl genes, respectively. ES cells into which the evening-getting vector has been appropriately introduced are cultured for the next stage.
  • An ES cell into which an evening-getting vector has been introduced is introduced into an early embryo derived from a different strain that has a distinct coat color from the strain from which the ES cell is derived. Generate as an animal. For example, in the case of a mouse, 129 strain-derived ES cells that have a gray hair color have various loci that have a black hair color and can be used as the best. It is desirable to use early embryos such as different C57BL / 6 mice. Thus, the chimera mouse can determine the chimera rate based on its hair color.
  • ES cells can be introduced into early embryos using the microinjection method (Hogan, B. et al. Manipulating the Mouse Embryo Cold Spring Habor Laboratory Press, 1988) or the aggregation method (Andra, N. et al. Proc. Natl. Acad. Sci. USA, 90, 8424-8428, 1993, Stephen, AW et al. Proc. Natl. Acad. Sci. USA, 90, 4582-4585, 1993) and the like.
  • the microinjection method uses ES cells from the 8-cell embryo to the blastocyst (blastocyst) This is a method of direct injection into the strike.
  • a recombinant embryonic stem cell is directly injected under a microscope into an embryo collected from an animal using a microphone-type manipulator or the like to produce a chimeric embryo. If this chimeric embryo is transplanted into the uterus of a foster parent (pseudopregnant animal) and developed, a desired chimeric animal can be obtained.
  • Chimera animals obtained from foster parents are further mated with wild-type animals of the same strain. About half of the resulting animals should have heterozygous Psil gene deleted chromosomes.
  • the dienotype of each individual can be determined temporarily by appearance characteristics such as hair color, and can also be determined by the above-described Southern blotting or dienotype analysis using the PCR method. Thus, once a heterozygous Psil gene-deficient animal is identified, the heterozygous Psfl gene-deficient animals can be crossed to obtain an animal having a Psil gene deficiency in the home.
  • Progeny of the Psfl gene-deficient animal produced as described above are also included in the Psfl gene-deficient animal of the present invention as long as the function of the Psil gene on the chromosome is deficient. 2.2 Use of Cre-loxP system
  • the loxP (locus of X-ing-over) sequence is a DNA sequence consisting of 34 base pairs and is a recognition sequence for Cre (Causes recombinat ion) recombination enzyme.
  • the two loxP sequences on the gene undergo specific recombination in the presence of the Cre protein.
  • the target gene to be deleted is replaced with a loxP sandwiched gene, and a Cre expression vector is incorporated, the target gene sandwiched between loxP is deleted due to the production of site-specific and time-specific Cre proteins. Can be made.
  • a targeting vector incorporating a marker gene such as a neomycin resistance gene sandwiched between the loxP gene on the 5 'side and the loxP gene on the 3' side of the Psfl gene region to be deleted according to 1) of the previous section 2.1).
  • a marker gene such as a neomycin resistance gene
  • ES cells can be selected by Southern blotting or PCR after selection by marker.
  • a Cre expression vector in which Cre protein is linked to a specific promoter is introduced into the homologous recombinant ES cells. From the obtained ES cells, an ES cell clone in which only the marker gene is deleted and the Psfl gene region is not deleted is identified by ⁇ recombination.
  • This ES cell is introduced into an animal according to 3) and 4) in the previous section 2.2 to obtain a Cre- ⁇ recombinant animal.
  • ⁇ -recombinant recombinant animals that have introduced a vector that has ⁇ genes at both ends of the Psf l 'gene and Cre-expressed recombinant animals that have introduced the Cre expression vector are prepared separately and mated. Depending on the situation, Cre-- ⁇ recombinant animals may be produced.
  • the Cre- Cre ⁇ recombinant animal thus obtained can be deficient in the Psfl gene in a site-specific and time-specific manner depending on the expression of the Cre protein. Therefore, it is extremely useful for functional analysis of the Psil gene at a specific time and at a specific site.
  • somatic cell clone is a clone generated by transplanting a nucleus extracted from a somatic cell into an enucleated unfertilized egg to produce a clone embryo, and then transplanting this cloned embryo into a foster mother's uterus.
  • a somatic cell clone is combined with a gene transfer technique, a desired recombinant animal clone can be obtained.
  • nuclei are removed from somatic cells that have been subjected to a recombination procedure that previously deletes the Psfl gene, and transplanted into enucleated unfertilized eggs to produce cloned embryos. If this cloned embryo is transferred to the uterus of a foster parent (pseudopregnant animal) to obtain a somatic cell cloned animal, this animal will have a Psil gene deficiency.
  • the Ps ⁇ gene-deficient animal of the present invention can be used as such a severe myelosuppression model. .
  • the model animal can be used for the development of drugs that restore myelosuppression (improving bone marrow function) and for screening such drugs.
  • Psi l gene-deficient animals are raised under test substance administration conditions and non-administration conditions, and changes in bone marrow function between the test substance administration conditions and non-administration conditions are determined by bone marrow MN C (mononuclear cells). Etc. are used as indicators.
  • a pile cancer drug such as 5-FU
  • the effect of the test substance as a bone marrow function improving agent can be evaluated.
  • hematopoietic stem cells are relatively increased in the bone marrow, so that it is possible to easily obtain a cell population containing hematopoietic stem cells at a high frequency without sorting the cells. Can do.
  • the resulting hematopoietic stem cells are useful for various experiments and drug screening.
  • the Psil gene is widely expressed in cells at the so-called stem cell level, and is predicted to control DNA replication in various stem cells. Therefore, the regulation of Psil genes—such as forced high expression of Psil genes—may induce stem cell self-renewal.
  • the Psf 1 gene is expressed in various stem cells including hematopoietic stem cells. It can also be applied to in vitro amplification technology for vesicles. In particular, in recent years, cancer is thought to be due to abnormalities in the stem cell system caused by cancer stem cells. By suppressing the function of the Psil gene, a method of inducing cancer stem cell death can also be developed.
  • the Psil gene-deficient animal of the present invention is useful for elucidating the function and action mechanism of Psil at the individual level. In order to elucidate the function at the individual level, in addition to the constant suppression of Psil expression, it is useful to suppress the expression of site-specific and time-specific Psil using the Cre-] oxP system described above.
  • Cdc45 is an essential protein for initiation of DNA replication, suggesting that Psfl has a similar function. Therefore, Psi protein and Psil gene can be used not only for the suppression of bone marrow function and treatment of diseases associated therewith, but also for the elucidation and treatment of DNA replication abnormalities and associated diseases.
  • bone marrow cells were purified with surface markers using FACS (Becton) and RT-PCR was performed in the same manner.
  • Differentiating antigens include Mac-1, Gr-1, Terll9, B220, CD4, I chose CD8. All differentiation antigens and specific antibodies against Sea-1 and c-kit were from Pharmingen.
  • the expression of Psil was also analyzed by RT-PCR in the leukemia cell line (BaF3) and melanoma cell line (B16).
  • Fig. 3 Paraffin-embedded sections of embryonic day 11 mice were prepared and immunostained using anti-Psfl antibodies.
  • Fig. 3 (A) anti-rabbit IgG antibody labeled with piotin was used as the secondary antibody, and ABC-HRP complex (manufactured by Dako) was used as the tertiary reagent. DAB (manufactured by Sigma) was used for color development.
  • Fig. 3 (B) an anti-rabbit IgG antibody labeled with Cy3 was used as the secondary antibody.
  • NIH3T3 cells were subjected to fluorescent immunostaining in the same manner as in FIG. 3 (B). Chromosomal DNA was stained with DAPI (Nacalai).
  • FIG. 2 shows the results of mouse Psfl expression analysis.
  • Psfl transcripts were found in bone marrow, thymus, testis, and ovary.
  • Differentiated and mature cells in bone marrow cells (Pii expression was not detected in Lii ⁇ , but undifferentiated cells (Lin + KIT ⁇ , Lin—KIT ⁇ Sca- ⁇ , LinllT ⁇ SCA-l 1 )
  • high expression of Psil was detected in the Lin IT ⁇ and Lin-KIT + Sca-1— cell groups, while Psfl expression was continually detected in tumor cell lines.
  • Figure 3 shows the localization of Psfl in fetal tissues.
  • Psil was expressed specifically in mesenchymal cells, AER region, heart and dorsal aorta.
  • Figure 4 shows the subcellular localization of Psfl in NIH3T3 cells.
  • the subcellular localization of Psfl varied depending on the cell cycle. In other words, Psil was localized in the nucleus during the interphase and in the cytoplasm during the M ′ phase.
  • Psfl gene-deficient mice were prepared by gene evening-targeting as follows. First, a genomic library of 129SV / J mice (Stratagene) was screened using the above mouse Psfl cDNA sequence (SEQ ID NO: 1) as a probe, and a plurality of mouse Psl genomic clones were identified. DNA was extracted from the resulting clone and subcloned into pBluescript l (Stratagene). Sequencing, restriction enzyme digestion, Southern blotting, etc. were performed to prepare a restriction enzyme map of the Psfl gene. Using the obtained Psfl genomic fragment, a targeting vector was prepared as follows.
  • the LacZ gene reporter; derived from pCMVb (Stratagene)
  • pGKneo positive selection marker; distributed by Kanazawa Univ., Masahide Asano
  • the short arm (5 'end region of intron 5)
  • LA2 (3' end region of intron 4) amplified in step 3 were connected to the 3 'and 5' sides, respectively.
  • a partial sequence of intron 4 and a diphtheria toxin fragment A gene (DT) as a negative selection marker were recombined on the 5 ′ side of LA2. With this targeting vector, almost all of exon 5 is replaced with LacZ_Neo cassette and approximately 20% of the amino acid is deleted (Fig. 5).
  • This evening-getting vector deletes the base sequence of 92781-92897 (exon 5) in the base sequence of mouse Psfl genomic DNA shown in GenBank Accession No. AL808125. Of the 196 amino acids, 39 amino acids will be deleted.
  • the prepared evening construction construct was introduced into ES cells derived from 129 mice by electroporation (E14.1; distributed by Kanazawa Univ., Asano), and contains G418 with mouse fibroblasts as feeder cells. Selection was performed with ES cell culture medium (GIBC0). The selected ES cells were further cultured and then subjected to DNA extraction according to a conventional method in order to identify homologous recombinants. The extracted DNA was subjected to Genotype screening by the above-mentioned Southern analysis. Southern plot analysis was performed using a labeled external probe (3 'probe) after digesting DNA with EcoRI. This external probe contains an outside of the homologous region used for the evening get vector. Exon 7 located at is used. Southern analysis revealed an approximately 7.5 kb band from the wild-type allele and an approximately 12.3 kb band from the mutant allele (Figure 6A).
  • ES cells in which homologous recombination was confirmed were aggregated with 8-cell embryos excised from C57B6 / J mice according to a conventional method to prepare chimeric embryos.
  • the chimeric embryo was established by transplanting the chimeric embryo into the uterine horn of a pseudopregnant ICR mouse, which was a foster parent.
  • the male mouse of the obtained mouse was mated with another female of wild type C57B6 / J mouse to obtain F1 mouse.
  • mice having a Psfl gene deficiency having a Psil gene deficiency in a heterozygote was selected from F1 mice, and these hetero mice were further mated.
  • Genotyping of mice was performed by PCR using DNA extracted from the mouse tail according to a conventional method. PCR conditions and the primers used are as follows. Wild Aril:
  • ReversPsf limer 5, -catcccagatcgttcttgttaacc-3 '(Guide II number 1 0)
  • Table 1 shows the results of genotyping.
  • the Psil gene-deficient animal of the present invention exhibits various symptoms caused by suppression of bone marrow function, and in particular, by administration of 5-FU or the like, symptoms similar to severe myelosuppression after administration of leukemia or anticancer drug are exhibited. . Therefore, it is useful for elucidating the pathology of such diseases, developing drugs, and screening drugs.
  • the PsiI gene-deficient animal of the present invention is useful as a source of hematopoietic stem cells. Sequence listing free text

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Abstract

L'invention décrit un mammifère non humain dont la fonction du gène Psf1 est absente et un procédé d’utilisation de celui-ci. À savoir, un mammifère non humain dont la fonction du gène Psf1 est absente sur son chromosome; l'utilisation de cet animal comme modèle animal pour des maladies d’aplasie médullaire; et un procédé de préparation de cellules souches hématopoïétiques et un procédé de dépistage d'un médicament utilisant cet animal.
PCT/JP2005/015496 2004-08-23 2005-08-19 ANIMAL DÉFICIENT EN GÈNE Psf1 ET PROCÉDÉ D'UTILISATION DE CELUI-CI WO2006022361A1 (fr)

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