WO2006022361A1

WO2006022361A1 - Psf1 GENE-DEFICIENT ANIMAL AND METHOD OF USING THE SAME

Info

Publication number: WO2006022361A1
Application number: PCT/JP2005/015496
Authority: WO
Inventors: Nobuyuki Takakura; Masaya Ueno
Original assignee: National University Corporation Kanazawa University
Priority date: 2004-08-23
Filing date: 2005-08-19
Publication date: 2006-03-02
Also published as: JP4590629B2; JP2006055110A

Abstract

A nonhuman mammal lacking the function of Psf1 gene and a method of using the same. Namely, a nonhuman mammal lacking the function of Psf1 gene on its chromosome; utilization of this animal as a model animal of bone marrow-depression diseases; and a method of preparing hematopoietic stem cells and a method of screening a drug using the animal.

Description

Specification

Psi l gene deficient animals and methods of use

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. In the late M phase, 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 After degradation, the cells transition from the G1 phase to the S phase. 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 ε. In studies using Saccharomyces cerevisiae, 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).

On the other hand, the existence of orthologs has been confirmed in mammals such as humans and mice. However, they were simply identified as homologues of yeast Psi l from sequence homology, and their function in the mammal has not been elucidated at all. Not only that, but also in yeast, the detailed functions of Psi l are almost unknown. It has not been. Disclosure of invention

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.

That is, the present invention relates to a non-human mammal deficient in the function of the Psfl gene on the chromosome (Psfl gene deficient animal).

In the 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.

1) Create a targeting vector for the deletion, substitution, and / or insertion of at least some sequences on the Psfl gene or its expression control region;

2) Introducing the vector into an ES cell to obtain an ES cell homologously recombined with the vector;

3) The recombinant ES cell is introduced into an early embryo and developed to obtain a chimeric animal;

4) 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.

As the Psil gene-deficient animal of the present invention, 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. Examples of such 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.

Since the Psfl gene animal of the present invention has suppressed bone marrow function, 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.

According to the present invention, 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. In addition, hematopoietic stem cells can be easily prepared using the Psfl gene-deficient animal of the present invention. 'Brief description of the drawings

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.

Hereinafter, the present invention will be described in detail.

1. Psfl gene deficient animals

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.

It has been reported that the Psil gene exists not only in yeast but also in mammals such as mice and humans. For example, the human Psil homolog is registered as KIAA0186 in Kazusa DNA Bank.

The 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)).

Even if an animal whose sequence is not registered in a public database, 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” according to the present invention includes all orthologs of the Psil gene as described above, and includes not only genomic DNA but also mRNA and cDNA. In the present invention, “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.

Since the Psfl genomic gene is long, a mutation that deletes or replaces a substantial part of its coding region can surely impair the function of the Psfl gene. The method for deleting the function of the Psfl gene will be described in detail in the next section.

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.

The “non-human mammal” according to the present invention is a mammal other than human. Although not particularly limited, rodents such as mice, rats, and rabbits are preferred.

Most preferred are mice in which ES cells are established and genetic recombination can be performed easily.

2. Production method of Psf l gene deficient animals

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. 2.1 Gene targeting

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).

1) Construction of one targeting vector

First, an evening-getting vector for deleting the Psi l gene is constructed. Prepare the genomic DNA library of the animal to be used prior to the construction of the getting vector. 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. In addition, a probe (external probe) for screening homologous recombinants is set outside the homologous region used for the targeting vector.

In the present invention, 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. For example, 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. In particular, mutations that delete or replace a substantial part of the exon region of the Psf gene In addition, the function of the Psi l gene can be deleted. In addition, 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. Examples of such 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 Α 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

Next, 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. Especially in mice, K14 strain, E14 strain, D3 strain, AB-1 strain, rabbit strain, R1 strain, TT2 derived from 129 mice Multiple cell lines, such as strains, are available. In mice, 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. For example, in the case of mouse ES cells, 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.

Introduction of the targeting vector into ES cells can be carried out by a known gene transfer method such as electoral positioning, microinjection, calcium phosphate method or the like. ES cells into which a targeting vector has been introduced can be easily selected with 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. In 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.

3) Production of chimeric animals

An ES cell into which an evening-getting vector has been introduced (recombinant ES cell) 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. In other words, 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.

On the other hand, in the Agrigesion method, one or two 8-cell embryos from which the zona pellucida has been removed and ES cells are co-cultured and aggregated to obtain chimeric embryos. If this chimeric embryo is transplanted into the uterus of a foster parent (pseudopregnant animal) and generated, a chimeric animal can be obtained. 4) Production of Psf l gene deficient animals

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

There is also a method for site-specific and time-specific deletion of target genes (Kulm R. et al., Science, 269, 1427-1429, 1995) using the Cre- オファー οχΡ system derived from bacteriophage P1 for gene targeting. is there. 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. In other words, if 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.

For example, 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). Create and introduce into ES cells. ES cells can be selected by Southern blotting or PCR after selection by marker. To confirm homologous recombination. 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 ΙοχΡ recombination. This ES cell is introduced into an animal according to 3) and 4) in the previous section 2.2 to obtain a Cre-ΙοχΡ recombinant animal. Alternatively, ΙοχΡ-recombinant recombinant animals that have introduced a vector that has ΙοχΡ 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--οχΡ recombinant animals may be produced.

The Cre- CreοχΡ 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.

2.3 Somatic cell clones

For animals where ES cells are not available, somatic cell clones (I. Wilmut et al, Nature, Vol. 385, 810-813, 1997, AE Schnieke et al, Science, Vol. 278, 2130-2133, 1997) It is also possible to produce Psil gene deficient animals. A 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. When this somatic cell clone is combined with a gene transfer technique, a desired recombinant animal clone can be obtained. That is, 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.

3. Phenotype of Psil gene deficient animals

In the Psi l gene-deficient animal of the present invention, when a phenotype different from that of the wild-type animal of the same litter appears, it is predicted that it is caused by the deficiency of the Psfl gene. For example, changes represented by the following were observed in mice lacking the Psi l gene.

1) Early lethality after implantation due to homodeficiency 2) Bone marrow suppression seen in heterozygous mice (Small 5-FU administration kills bone marrow function and is fatal)

4. Use of Psil gene deficient animals

1) Myelosuppression model animal

Conventionally, a means of suppressing bone marrow function by irradiation with a lethal dose of radiation has generally been used for hematopoietic stem cell transplantation, but in a Psil gene-deficient mouse of the present invention, a small amount of 5-FU was administered to suppress the bone marrow function. Therefore, it can be used in an experimental system for observing the hematopoietic function without irradiation.

In some cases, leukemia and anti-cancer drugs are administered, but bone marrow suppression does not recover and death occurs. Since the situation after 5-FU administration to Psi l gene-deficient mice is similar to these cases, the Ps Π 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.

For example, 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. Alternatively, by co-administering the test substance with a pile cancer drug such as 5-FU, and similarly evaluating the change in animal survival rate and bone marrow function under the test substance administration condition and non-administration condition The effect of the test substance as a bone marrow function improving agent can be evaluated.

2) Preparation of hematopoietic stem cells

When 5-FIJ is administered to the Psf 1-deficient mouse of the present invention, 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.

3) In vitro proliferation of stem cells

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. In other words, 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.

4) Other

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.

Psfl gene-deficient embryos showed similar phenotypic changes to Cdc45-deficient embryos. 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. Example '

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Example 1] Expression analysis of Psil

1. Test method

(1) Expression analysis of mouse Psfl by RT-PCR

Total RNA was extracted from various mouse tissues (brain, heart, lung, thymus, liver, spleen, bone marrow, testis, ovary) and cDNA was extracted using Superscriptll reverse transcriptase and oligod (T) (both from Invitrogen). Synthesized. PCR was performed using ExTaQ DNA polymerase (manufactured by TaKaRa) with this cDNA as a saddle. 5 '-acc acagtccat gcc ate ac-3' (SEQ ID NO: 3) and 5, -tec acc acc ctg ttg ctg ta-3 '(SEQ ID NO: 4) for GAPDH amplification, 5' for Psfl amplification -tta aga aat aga cgc tgc acg a-3 '(SEQ ID NO: 5) and 5'-tgc cat cat caa ctt caa att c-3' (SEQ ID NO: 6) were used as primers.

Similarly, bone marrow cells were purified with surface markers using FACS (Becton) and RT-PCR was performed in the same manner. Differentiating antigens (Lin) 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).

(2) Analysis of Psfl localization in fetal tissues by immunostaining

Paraffin-embedded sections of embryonic day 11 mice were prepared and immunostained using anti-Psfl antibodies. In the experiment of 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. In Fig. 3 (B), an anti-rabbit IgG antibody labeled with Cy3 was used as the secondary antibody.

(3) Subcellular localization of Psil (Relationship with cell cycle)

NIH3T3 cells were subjected to fluorescent immunostaining in the same manner as in FIG. 3 (B). Chromosomal DNA was stained with DAPI (Nacalai).

2. Test results

(1) Results of mouse Psfl expression analysis

Figure 2 shows the results of mouse Psfl expression analysis. In adult mouse tissues, 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- Γ, LinllT ^ SCA-l ¹ ) In particular, 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.

(2) Localization of mouse Psfl in tissue

Figure 3 shows the localization of Psfl in fetal tissues. In fetal tissues, Psil was expressed specifically in mesenchymal cells, AER region, heart and dorsal aorta.

(3) Intracellular localization of mouse Psil (Relationship with cell cycle)

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.

[Example 2] Preparation of Psfl gene-deficient mice 1. Test method

(1) Generation of Psf l gene-deficient mice

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)) and pGKneo (positive selection marker; distributed by Kanazawa Univ., Masahide Asano) were linked to the LacZ-Neo cassette and the resulting genomic fragment was PCR-processed in a saddle type. The short arm (5 'end region of intron 5) and LA2 (3' end region of intron 4) amplified in step 3 were connected to the 3 'and 5' sides, respectively. Furthermore, 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. Next, the male mouse of the obtained mouse was mated with another female of wild type C57B6 / J mouse to obtain F1 mouse.

Furthermore, a mouse 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:

Forward primer: 5 '-ggaattcggccccccaaaagcctatatat-3' (SEQ ID NO: 7) ReversPsf limer: 5 '-catcccagatcgttcttgttaacc-3' (layout [J number 8) Mutant aryl:

Forward primer: 5, -ccgaacgcgtataacttcgtaiagc-3 '(Care! 1 number 9)

ReversPsf limer: 5, -catcccagatcgttcttgttaacc-3 '(Guide II number 1 0)

2. Test results

Table 1 shows the results of genotyping.

〔table 1〕

Age No. ofoffspring with genotype: _{resprbed † pta |} neonate. 9 14 0 0 23

E11.5 9 14 0 3.26 '

E9.5 7 16 0 6 • 29

E7.5 12 16 0 4 32

E6.5 ND ND ND 4 20

E3.5 4 13 5 NA 22 As is clear from Table 1, embryos that are homozygous for the Psf 1 gene are prematurely lethal after implantation (ND; not determine (determined by morphological findings), NA; not available) ^ Figure 6B ( E6.5) in the table shows tissue slices of Psil gene homo-deficient (_ /-) embryos and wild-type embryos. Homo-deficient (-/-) embryos clearly showed abnormal morphology.

[Example 3] Comparison of Psil gene homo-deficient (-/-) embryo and hetero-deficient (+/-) embryo 1. Test method

(1) In vitro culture of blast cysts (Fig. 7)

On day 3.5 of gestation, return the uterus and collect blast cysts. This was cultured in ES medium for 6 days on a plastic dish coated with 0.1% gelatin. In the uptake experiment of BrdU (5-bromo-2 'deoxy-uridine) (manufactured by Sigma), BrdU was added to the medium to a final concentration of lOmM and cultured for 12 hours. Anti-BrdU antibody (manufactured by Zymed) was used to detect the incorporated BrdU. 2. Test results

When blast cysts of wild type or heterozygous Psil gene are cultured, ICM (inner cell mass) divides vigorously and trophoblasts adhere to the surface of the culture dish and proliferate. Cysts did not show vigorous cell division of ICM (Fig. 7). Furthermore, ICM BrdU incorporation was hardly detected (Fig. 8). This revealed that Psil is essential for cell division in early embryos.

[Example 4] Comparison of Psil gene deficient (+/-) mice and wild type mice

1. Test method

150 ng / kg of 5-FU (manufactured by Sigma) was intravenously administered to the Psil gene-deficient (+/-) and wild-type mice (6 weeks old, N = 5 each) prepared in Example 2 Compared. The results are shown in FIG. 9 (A: survival, B: blood MNC, C: bone marrow MNC) and FIG. 2. Test results (1) Survival rate

All wild-type mice were alive, but all PsiI-deficient mice died within one week. Half of the wild-type mice survived even when 350 mg / kg (corresponding to LD50) of 5-FU was administered.

(2) Blood MN C and bone marrow MN C

There was no significant difference in blood MN C changes between the two mice, but bone marrow MN C showed recovery in wild-type mice, but no recovery in Psi l gene-deficient mice. It was.

(3) Analysis of bone marrow cells

The morphology of whole bone marrow cells of Psi l gene deficient (+/-) mice and wild type mice was analyzed by FACS. In Psil gene hetero-deficient mice, there was almost no increase in cell populations with high FSC (white blood cells such as lymphocytes, monocytes, granulocytes) after 5-FU administration. In wild type, Lin (except Mac-l) —Sea-1¾IT ^{ hematopoietic stem cells become moderately expressed Mac-1 after 5-FU treatment, but Psi l gene heterodeficient mice There was almost no appearance of dividing stem cells expressing Mac-1. 5- Day 5 after FU administration Analyzing the DNA content of the stem cell population (Lin (excluding Mac-1) —Sea-1 IT ^÷ ), DNA synthesis in most cells in Psf l gene heterodeficient mice It was found that the project was not carried out and remained in the G1 period.

As described above, Psi l gene hetero-deficient mice showed various phenotypic changes that may be caused by bone marrow dysfunction. All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety. Industrial applicability

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

SEQ ID NO: 3—Description of artificial sequence: Primer SEQ ID NO: 4 Description of one artificial sequence: Primer SEQ ID NO: 5—Description of artificial sequence: Primer SEQ ID NO: 6—Description of artificial sequence: Primer SEQ ID NO: 7—Description of artificial sequence: Primer SEQ ID NO: 8—Description of artificial sequence: Primer SEQ ID NO: 9 Description of one artificial sequence: Primer SEQ ID NO: 1 0—Description of artificial sequence: Primer

Claims

1. A non-human mammal deficient in the function of the Psfl gene on the chromosome.

2. The function of the Psf l gene is deficient in one allele on the chromosome by deletion, substitution of at least a portion of the Psf l gene or its expression control region, and insertion of Z or other sequences. The non-human mammal according to claim 1. Mouth blue

3. The non-human mammal according to claim 1 or 2, produced by the following steps: 1) lack of at least a part of the sequence on the Psfl gene or its expression control region

Create targeting vectors for deletion, substitution, and insertion of Z or other sequences;

2) Introducing the above vector into ES cells to obtain ES cells homologously recombined within the vector range;

4) Mating the F1 heterozygous animals obtained by mating the above chimeric animals with wild-type animals, and obtaining the animal lacking the function of the Psfl gene on the chromosome from the F2 animals obtained or their progeny. 4. The non-human mammal according to any one of claims 1 to 3, wherein the non-human mammal is a mouse.

5. The non-human mammal according to any one of claims 1 to 4, which is a myelosuppression model animal.

6. A method of obtaining a hematopoietic stem cell from the bone marrow of a non-human mammal according to any one of claims 1 to 5 by administering a myelosuppressive agent to the non-human mammal.

7. A hematopoietic stem cell of a Psfl gene-deficient non-human mammal obtained by the method according to claim 6.

8. A screening method for a bone marrow function improving agent using the non-human mammal according to any one of claims 1 to 5.

9. A method of amplifying the stem cell in vitro by forcibly expressing the Psfl gene in the stem cell.