WO2004067744A1

WO2004067744A1 - Self-replication determining factor of embryonic stem cell

Info

Publication number: WO2004067744A1
Application number: PCT/JP2004/000790
Authority: WO
Inventors: Shinya Yamanaka
Original assignee: Sumitomo Pharmaceuticals Co., Ltd.
Priority date: 2003-01-31
Filing date: 2004-01-28
Publication date: 2004-08-12
Also published as: JPWO2004067744A1; JP4268169B2

Abstract

It is intended to provide an ES cell with forced expression of ECAT4. The ES cell with forced expression of ECAT4 can be cultured in the absence of feeder cells or LIF. It is also intended to provide a consensus sequence recognized by ECAT4, an ECAT4 enhancer, an ECAT4 promoter region and use thereof. Since it is clearly indicated that ECAT4 has a function as a transcriptional factor promoting self-replication of ES cells, it is highly useful in research and development of ES cells.

Description

Specification

Determinants of self-renewal of embryonic stem cells

The present invention relates to a mammalian cell in which ECAT4 is forcibly expressed, an ECAT4 enhancer, a consensus sequence recognized by ECAT4, and uses thereof. Background art

Embryonic stem cells (ES cells), derived from the inner cell population of the mammalian blastula, grow rapidly and indefinitely, while retaining the pluripotency, or the ability to differentiate into many types of cells. This property of ES cells is maintained by symmetric self-renewal, which results in two identical stem cell daughter cells during cell division. From these two properties, the potential of a new regenerative medicine that uses ES cells to create tissues and cells and use them for treatment is attracting attention. However, human ES cells also have ethical issues because human embryos must be destroyed for the production of ES cells.

One solution to avoid such ethical issues is to produce pluripotent cells directly from somatic cells and other cells. The first step toward this goal is the identification of a master gene that functions specifically in pluripotent cells and determines the characteristics of the cells.

To date, two transcription factors, STAT 3 and Oct 3/4, have been shown to be important for the self-renewal of mouse ES cells (Burdon et al., (1999). Tissues Organs 165, 131-43., Niwa, (2001). Cell Struct Funct 26, 137-48.

STAT 3 is activated by leukemia inhibitory factor (LIF), a type of cytokine essential for maintaining pluripotency (Smith et al., (1988). Nature 336, 688-90., Williams et al., (1988). Nature 336, 684-7.). Activated STAT 3 alone supports self-renewal in the absence of LIF (Matsuda et al., (1999). Embo J 18, 4261-9.). Even overnight gene-getting experiments have shown the importance of STAT 3 for self-renewal of ES cells (Raz et al., (1999). Proc Natl Acad Sci USA 96, 2846-51.). However, STAT3 expression is widespread. It is found in a range of cell types and induces differentiation in some cells (Nakajima et al., (1996). Embo J 15, 3651-8.).

Another transcription factor, Oct 3/4, considered to be the key to ES cell self-renewal, is a POU family transcription factor that is expressed in pluripotent cells such as ES cells, ectoderm and primitive embryonic cells I do. Disruption of the mouse Oct 3/4 gene resulted in death of the early embryo shortly after implantation, and the inner cell mass of the blastula lacking Oct 3/4 was not pluripotent and became trophoblast lines. Only differentiate. Niwa eta1 confirmed this in ES cells and showed that suppressing Oct 3/4 resulted in autonomous differentiation into trophoblasts (Niwa et al., (2000). Genet 24, 372-6.). Combining these data with its specific expression pattern suggests that Oct 3/4 may be the master gene for pluripotent cells.

However, ES cells that constitutively express Oct 3/4 from an exogenous promoter still require LIF for self-renewal, even when the appropriate amount of 〇ct 3/4 is expressed. Removal of LIF results in differentiation to primitive endoderm (Niwa et al., (2000). Nat Genet 24, 372-6.). Disclosure of the invention

An object of the present invention is to provide a cell in which ECAT4 is forcibly expressed, an ECAT4 enhancer, a consensus sequence recognized by ECAT4, and uses thereof.

Activating the transcription or function of key self-renewal determinants for ES cells can be expected to improve the efficiency and economy of ES cell culture. In addition, it can be expected that undifferentiated cells can be reliably eliminated when ES cells are induced to differentiate into specific functional cells by suppressing the transcription or function of the factor. The risk of appearance of tumor cells (teratomas) derived from keratinocytes can be avoided. Therefore, a major self-renewal determinant is found for ES cells, and an enhancer that regulates the transcription or function of the factor is used to search for a substance capable of inducing or inhibiting the differentiation of ES cells, Very useful for research and development.

The present inventors have already found the ECAT4 gene as a gene specifically expressed in ES cells. are doing. Intensive studies of the ECAT4 gene show that targeted disruption of the mouse ECAT4 gene in ES cells results in a spontaneous differentiation that results in an extraembryonic endoderm lineage that expresses high levels of endodermal markers. confirmed. The ECAT4-deficient blastocysts died shortly after implantation, and the inner cell mass of the ECAT4-deficient blastocysts was in vitro and hardly proliferated. We also found that ES cells in which ES cells had the ECAT4 gene forcibly expressed maintain undifferentiated state even when cultured in the absence of LIF, so ECAT4 is a major ES cell. It was found to be an important self-replicating determinant.

In addition, the inventors have identified a consensus sequence to which ECAT enhancer and ECAT4 bind. In addition, the EC AT 4 consensus sequence to which the EC AT 4 protein binds is located 4 kb upstream of the transcription initiation site of the 5′-flanking region of the mouse GAT A 6 gene. It was confirmed that this region was well conserved in the gene sequences of the six genes.

The present invention has been completed based on these findings.

That is, the gist of the present invention is as follows.

Item 1. Self-renewal determinant of embryonic stem (ES) cells consisting of ECAT4,

Item 2. The ES cell self-renewal determinant according to Item 1, wherein ECAT4 is mouse ECAT4, human ECAT4 or monkey ECAT4,

Item 3. A monkey EC AT4 gene containing a DNA described in the following (a) or (b): (a) a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 10,

(b) a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 10 under stringent conditions and has an ability as a self-renewal determinant of ES cells

NA,

Item 4. A mammalian stem cell in which ECAT4 is forcibly expressed,

Item 5. The mammalian stem cell according to Item 4, wherein the stem cell is an ES cell,

Item 6. The cell according to Item 4 or 5, wherein ECAT4 is mouse ECAT4, human ECAT4 or monkey ECAT4.

Item 7. An amplifier containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide represented by SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 or a part thereof Chisense polynucleotide,

Item 8. A cell differentiation promoting agent comprising the antisense polynucleotide according to Item 7, Item 9. ECAT4 enhancer,

Item 10. The ECAT4 enhancer according to Item 9, comprising a part or all of the nucleotide sequence represented by SEQ ID NO: 7.

Item 11. The ECAT4 enhancer according to Item 9, which has the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

Item 12. The ECAT 4 enhancer according to Item 11, which is a part of the nucleotide sequence represented by SEQ ID NO: 15, and comprises the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 8 or SEQ ID NO: 19,

Item 13. The ECAT4 enhancer according to Item 9, which contains the nucleotide sequence represented by SEQ ID NO: 15.

Item 14. The ECAT4 enhancer according to Item 13, which is a part of the nucleotide sequence represented by SEQ ID NO: 14, and contains the nucleotide sequence represented by SEQ ID NO: 15.

Item 15. The base sequence of ECAT4 enhancer according to any one of Items 10 to 14, which comprises a base sequence in which one or more bases have been deleted, substituted or added, and the ECAT4 gene ECAT4 sensor according to item 9, which has an ability to promote transcription of

Item 16. Consisting of the nucleotide sequence of ECAT4 enhancer according to any one of Items 10 to 14: hybridizes with DNA under stringent conditions and has an ability to promote transcription of ECAT4 gene EC AT 4 Enhancer according to item 9, which is characterized in that:

Item 17. An EC A4 promoter region comprising the EC AT 4 enhancer according to any one of Items 9 to 16.

Item 18. Consensus sequence recognized by ECAT4,

Item 19. The consensus sequence according to Item 18, wherein the consensus sequence comprises a DNA described in any of the following (a) to (c):

(a) a DNA consisting of the base sequence represented by SEQ ID NO: 4,

(b) one or more bases are deleted in the base sequence represented by SEQ ID NO: 4, A DNA consisting of a substituted or added base sequence and capable of binding to ECAT4;

(c) a DNA having the ability to hybridize with a DNA consisting of the nucleotide sequence of SEQ ID NO: 4 under stringent conditions and bind to ECAT4;

Item 20. The consensus sequence according to Item 18, comprising a DNA described in any of the following (a) to (c):

(a) a DNA consisting of the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 16,

(b) consisting of a nucleotide sequence in which one or more nucleotides have been deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 16, and

DNA having the ability to bind to E CAT 4,

(c) a DNA having the ability to hybridize with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 16 under stringent conditions, and to bind to ECAT4;

Item 21. A recombinant vector characterized by containing the enhancer according to any one of Items 9 to 16, the promoter region according to Item 17, or the consensus sequence according to any one of Items 18 to 20,

Item 22. A transformed cell transformed with the recombinant vector according to Item 21,

Item 23. A method for screening a substance having an activity of controlling the activity of an ECAT4 enhancer or ECAT4 promoter region, comprising the following steps (a), (b) and (c):

() A step of contacting a test substance with a cell transformed with a DNA obtained by linking the ECAT4 promoter region according to any one of Items 9 to 16 or the ECAT4 promoter region according to Item 17 to a reporter gene,

(b) measuring the activity of the repo overnight gene in cells contacted with the test substance, and comparing the expression level with the activity in control cells not contacted with the test substance;

(c) selecting a test substance that controls the activity of the ECAT4 enhancer or ECAT4 promoter region based on the comparison result of the above (b),

Item 24. ES cells transfected with p CAG-IP vector into which ECAT4 gene has been inserted Item 25. A genetically modified animal obtained by injecting the ES cell according to Item 24. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the identification of ECAT4.

(A) Comparison of mouse Nkx2.3, mouse ECAT4 and its homologous protein FL J12581. (B) RT—PCR analysis showing expression profiles of mouse ECAT4, Oct 3/4 and NAT1. 1. Undifferentiated MG1. 19 ES cells; 2. Differentiated MG1. 19 ES cells; 3. Undifferentiated RF8 ES cells (RT_ minus control); 4. Undifferentiated RF8 ES cells; 5. Differentiated MG1. 19 7. Heart; 8. Kidney; 9. Testis; 10. Spleen; 11. Muscle; 12. Lung; 13. Stomach; 14. Ovary; 15. Thymus; 16. Liver; . (C) RF8, Jl, 008] \\ 401. Estamplot analysis showing ECAT4 expression in the 19 ES cell line. Extracts from ES cells kept in the undifferentiated state (U) or treated with RA for 5 days (D) were analyzed. Western plots were performed using anti-ECAT4 serum or anti-CDK4 antibody.

FIG. 2 shows the targeted disruption of the mouse ECAT4 gene.

(A) Mouse ECAT4 gene, evening targeting vector, and gene structure after homologous recombination. The position of the probe and restriction enzyme used for Southern blot is shown. Arrows indicate primers for PCR analysis. (B) Southern blot using 3 'single probe shows 11 kb band from wild type locus, 15.4 kb band from 3-geo target locus, and A 3 kb band resulted. (C) PCR yields a 3.2 kb band from the wild-type locus, an 8.5 kb band from the / 3—geo target locus, and a 4.2 kb band from the hygr target locus. Was. (D) Northern blot confirmed that no transcript of ECAT4 was present in the knockout cells.

FIG. 3 shows an analysis of ECAT4 deficient ES cells.

(A) Morphology of wild-type RF 8 cells grown on STO feeder cells (top). EC AT 4 cells (middle) grown on STO support cells and EC AT grown without support cells 4 Deficient cells (bottom). (B) Growth curve. (C) Northern plot analysis for marker genes. (D) RT-PCR analysis of the marker gene.

Figure 4 shows the analysis of pre-implantation embryos.

(A) Eight-cell embryos and blastocysts obtained from wild-type females and heterozygotes were stained with X-gal. (B) Blastocysts obtained by heterozygous crossing were grown on gelatin-coated dishes using ES cell culture media. Morphology was recorded 10 days later and genotype was determined by PCR.

FIG. 5 shows the forced expression of ECAT4 from the constituent promoters. (A) PCR-RT analysis showing expression levels of ECAT4, Oct 3Z4 and NAT1 in MG1.19 cells transformed with parental plasmid or CAG-ECAT4. Cells were grown in the presence or absence of LIF. (B) Western plot confirmed constitutive expression of exogenous ECAT4. (C) Morphology of control (left) and CAG-ECAT4 expressing cells (right) grown in the presence (top) or absence (bottom) of LIF. (D) Cell growth rate

FIG. 6 shows the transcriptional regulation of ECAT4.

Sequence logo showing consensus sequence for ECAT4 binding obtained by SELEX (Schneider and Stephens, 1990). The generation of the sequence logo was completed using WebLogo (www.bio.cam.ac.uk/cgi—bin/seqlogo/logo.cgi). (B) ECAT4 consensus sequence found in the 5 'regulatory region of mouse and human GATA-6. A luciferase repo overnight gene containing this region was constructed.

FIG. 7 relates to the Enhansa single region analysis of the mouse ECAT4 gene.

(A) Mouse ECAT4 gene structure and DNA fragment used for enhancer analysis. (B) Repo overnight gene structure used for Enhancer analysis. The gene fragments shown in Fig. 7A were inserted into the three ends of the Levo Yuichi gene consisting of the promoter of the thymidine kinase gene, luciferase cDNA and poly-A addition sequence.

FIG. 8 relates to the Enhansa single region analysis of the mouse ECAT4 gene.

(A) The region of the fragment used for the Enhancer analysis (left) and the activity of the fragment (right)

) o (B) Enhansa region (region of 4737 / —4387) of the mouse ECAT4 gene. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, in the present specification, the abbreviations of amino acids, (poly) peptides, (poly) nucleotides and the like are referred to by the rules of IUPAC-IUB. CIUPAC-IUB Coanda union on Biological Nomenclature, Eur. (1984)], "Guidelines for Preparation of Specifications Containing Base Sequences or Amino Acid Sequences" (edited by the Japan Patent Office) and conventional symbols in the art.

The term "ES cell self-renewal determinant" of the present invention refers to a master gene involved in the self-renewal ability of embryonic stem cells (ES cells) and the like to replicate the same cell without differentiation. The determinant of self-renewal of ES cells can be used as a marker for cells having self-renewal ability such as ES cells. By examining the expression of this factor, it is determined whether the examined cell has self-renewal ability. Alternatively, it can be tested for ES cells. By forcibly expressing this factor, cell proliferation can be safely maintained in the absence of feeder cells and in the absence of cytokines such as IF, while maintaining the totipotency of ES cells. This can be expected to improve the efficiency and economics of ES cell culture. In addition, since there is no risk of contamination with infectious substances such as endogenous virus derived from feeder cells, which is a concern when clinically applied, improved safety can be expected.

When ES cells are induced to differentiate into specific functional cells, known methods such as homologous recombination and antisense RNAi (RNA inhibitors) (Experimental method for gene function inhibition by Kazu Makoto Tahira, Yodosha, 2001) By suppressing the expression of the self-renewal determinant of ES cells using), undifferentiated cells can be expected to be eliminated more reliably than before. As a result, in clinical applications, safety can be expected to be further improved by avoiding the danger of the emergence of undifferentiated cell-derived tumor cells (teratoma), which is a concern.

The “ECAT4” of the present invention is a natural ECAT4 or a recombinant ECAT4, and may be of mammalian origin. For example, mouse ECAT4, rat ECAT4, monkey ECAT4 ゃ human ECAT4 and the like can be mentioned.

The “gene” or “DNA” has a specific nucleotide sequence (SEQ ID NO: 4 to: L 0 14 Not only the `` gene '' or `` MA '' shown in 19), but the biological function is equivalent as long as the function (activity) is the same, or in the case of a gene encoding a protein To the extent possible, it includes “genes” or “DNAs” that encode the congeners (homologs), derivatives and variants thereof. The “gene” or “DNA” encoding these homologs (homologs), derivatives and mutants is specifically shown under any of stringent conditions under any of the aforementioned SEQ ID NOs: 410. "Gene" or "DNA" having a base sequence that hybridizes with a specific base sequence.

Stringent conditions include nucleic acid binding to a complex or probe, as described in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). Can be determined based on the melting temperature (Tm). For example, as washing conditions after the hybridization, conditions of about “1XSSC 0.1% SDS 37 ° C.” can be usually mentioned. The complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions. Although not particularly limited, more severe hybridization conditions can be about 0.5XSSC 0.1% SDS 42 ° C, and more severe hybridization conditions can be about 0.1XSSC, 0.1% SDS, 65 ° (). Specifically, as such a complementary strand, a strand consisting of a base sequence completely complementary to the base sequence of the target positive strand, and 70% or more, preferably 80% or more, More preferably, a chain consisting of a base sequence having homology of 90% or more, more preferably 95% or more can be exemplified.

In addition, a gene encoding a homologue (homolog) of the specific base sequence (SEQ ID NO: 4 10 14 L9), for example, a gene of another species corresponding to a human gene is Horn oloGene (http :, ncbi. It can be identified by nlm. nih. gov / HomoloGene /). Specifically, the specific human nucleotide sequence is subjected to BLAST (Proc. Natl. Acad. Sci. USA., 90: 5873-5877, 1993 http://ww.ncbi.nlm.nih.gov/BLAST/) to be matched. (Score is highest, E-value is 0 and Identity shows 100%) Acquisition number of sequence and other species gene obtained by inputting the accession number to Homo loGene Can be selected from a list showing the correlation of gene homologues with human genes. Wear. The gene or DNA does not matter whether it is a functional region, and may include, for example, an expression control region, a coding region, an exon or an intron.

In the present specification, when a term such as “E CAT 4 gene” or “£ 〇8 cho 4 1)” is used, unless otherwise specified, the EC AT 4 gene (DNA) represented by the specific nucleotide sequence (SEQ ID NO: 8-10) are used for the purpose of including genes (DNA) encoding their homologs, mutants and derivatives. Specifically, the human ECAT4 gene described in SEQ ID NO: 9 (GenBank Accession NO. AB093576), the mouse ECAT4 gene described in SEQ ID NO: 8 (GenBank Accession NO. AB093574), and the SEQ ID NO: 10 The described monkey EC AT4 gene and a rat homolog are included.

As used herein, the term “polynucleotide” is used to include both RNA and DNA. The DM includes any of cDNA, genomic DNA and synthetic DNA. The above-mentioned RNA includes total fragile, mRNA and synthetic RNA.

In the present specification, “protein” or “(poly) peptide” includes not only “protein” or “(poly) peptide” represented by a specific amino acid sequence (SEQ ID NOs: 1 to 3), but also Fragments, homologs (homologous splice variants), variants, derivatives, amino acid modifications, etc., are included as long as their biological functions are equivalent. Here, examples of homologues include proteins of other species such as mouse and rat corresponding to human proteins, and these are obtained by 11011101 (^ 611601 0: 〃 ^ .1 ^ bi.nlm.nih.gov/HomoloGene/). It can be determined a priori from the nucleotide sequence of the identified gene. Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having an amino acid sequence altered by artificial deletion, substitution, addition and insertion. You. In addition, examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homologous to the protein or (poly) peptide having no mutation. Amino acid modifications include naturally occurring amino acids and non-naturally occurring amino acids, and specifically include phosphorylated amino acids. Therefore, when the term “ECAT4 protein” or simply “ECAT4” is used herein, unless otherwise specified, the specific amino acid sequence (SEQ ID NO: 1) To 3), and their congeners, variants, derivatives, amino acid modifications and the like. Specifically, human ECAT 4 having the amino acid sequence of SEQ ID NO: 2, mouse ECAT 4 having the amino acid sequence of SEQ ID NO: 1, and amino acid sequence of SEQ ID NO: 3 Monkey ECAT4, and their rat homologs.

Hereinafter, the specific contents of the present invention will be described.

(1) Mammalian stem cells in which EC AT 4 is forcibly expressed

The “mammalian stem cell in which ECAT4 is forcibly expressed” of the present invention is a cell obtained by forcibly expressing ECAT4 in a mammalian cell. Mammalian cells are cells derived from tissues, organs, etc. of mammals such as humans, monkeys, mice, rats, etc., and may be primary cells taken from individuals, cultured cells, or mammalian cells. Any animal-derived cells may be used. Mammalian cells include, for example, commercially available cultured cells (such as ATCC), mouse ES cells, monkey ES cells ゃ human ES cells, and more preferably, mouse ES cells, monkeys that differentiate in the absence of IF. ES cells ゃ embryonic stem cells such as human ES cells, but are not limited thereto.

For forced expression of ECAT4, for example, an expression vector containing the ECAT4 gene may be introduced into cells. Specific methods for introducing an expression vector include the calcium phosphate method, DEAE-dextran, and the like. And known methods such as electroporation, a method using a lipid for transfection (Lipofectamine, Lipofectin; Gibco-BRL), and a microinjection method.

An expression vector containing the ECAT4 gene can be produced based on ordinary genetic engineering techniques. The expression vector used here can be appropriately selected depending on the host used, the purpose, and the like, and examples thereof include a plasmid, a phage vector, and a virus vector.

For example, plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.K pRc / RSV and pRc / CMV, and viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors.

The vector may optionally have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, and a terminator. Further, it may have a gene encoding a fusion protein of the marker overnight protein and ECAT4.

Here, the “ma-chi-ichi-gene” refers to a gene whose phenotype is clear and is useful for genetic analysis, and the expression of which can be directly observed when the gene is introduced. Examples include fluorescent protein genes such as CFP and YFP, which are mutants of GFP and GFP, but are not limited thereto. All that can confirm that the gene has been introduced in the above are included in the category of the marker-gene of the present invention. In addition, a protein is a protein encoded by a marker gene.

By introducing a vector to which such a marker gene is added, cells expressing ECAT 4 can be easily detected. Furthermore, by using ES cells into which such a gene has been introduced, only stem cells that express ECAT4 can be selected. Thus, when ES cells are divided into functional cells, undifferentiated cells and differentiated cells can be selected. It can also be used as a marker that can select This can avoid the danger of transplanting undifferentiated cells into a living body, which may be at risk of teratoma formation. Furthermore, an expression vector containing the ECAT4 gene can also be produced using a conditional gene expression control system. For example, a forced expression system in which the introduced gene of interest is expressed in the presence of tetracycline but not in the absence of tetracycline can be mentioned (Niwa et al., (2000). Nat Genet 24, 372-6.). Cells into which these vectors have been introduced can switch on and off ECAT4 expressing cells depending on the culture conditions, so that ECAT4 is stably forcibly expressed between ES cells, and expression is induced when differentiation is induced. It is thought that differentiation can be efficiently induced by shutting off, and can be used for various purposes.

The expression vector containing the ECAT4 gene may be a vector having the property of disappearing under certain conditions. Specifically, for example, a pCAG-IP vector and the like can be mentioned. ES cells into which the ECAT4 expression vector having such properties has been introduced are very useful for producing genetically modified animals such as transgenic mice. In the ES cells into which the vector has been introduced, the vector itself disappears from the cell under certain conditions. Genetically modified animals can be prepared more simply and reliably than controlling the expression level of ECAT4.

For example, CAG-EC AT cells produced using a pCAG-IP vector containing the ECAT 4 gene can be cultured in the absence of LIF ゃ feeder cells. The operation of introducing another vector for expressing, knocking out, knocking down or knocking in the desired target gene into the target gene is very easy.

Here, the genetically modified animal is an animal in which the standard gene is overexpressed, knocked out, knocked down or knocked in. The genetically modified animal can be prepared by a known method, and the animal may be a mammal, and examples thereof include a mouse and a rat. ,

(2) Antisense polynucleotide of the present invention

The antisense polynucleotide of the present invention has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the ECAT4 gene or a part thereof, and can suppress the expression of the ECAT4 gene. Any substance having an action may be used, and examples thereof include antisense RNA and antisense DNA.

Here, the substantially complementary nucleotide sequence is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more of the nucleotide sequence complementary to the nucleotide sequence of the ECAT4 gene. % Or more, most preferably about 95% or more homology. In particular, about 70% or more, preferably about 80% or more, of the complementary sequence of the base sequence (for example, the base sequence near the start codon) of the portion encoding the N-terminal portion of the base sequence of the ECAT4 gene More preferably, an antisense polynucleotide having about 90% or more, most preferably about 95% or more homology is suitable. Specific examples include a sequence complementary to or substantially complementary to the base sequence described in any of SEQ ID NOs: 8 to 10, or an antisense polynucleotide having a part thereof. A polynucleotide having a complementary base sequence (complementary chain, reverse chain) refers to the full-length sequence of a polynucleotide consisting of the base sequence shown in each of the above-mentioned sequence numbers, or at least 15 continuous bases in the base sequence. For the partial sequence having the base sequence (for convenience, these are also referred to as “positive chains”), base pairing such as A: T and G: C 1.4 means polynucleotides that are in a complementary relationship with each other. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the base sequence of the target positive strand, but has a complementary relationship sufficient to hybridize with the target positive strand under stringent conditions. May be provided.

In addition, the polynucleotides on the positive chain side include not only those having the base sequence of the ECAT4 gene or a partial sequence thereof, but also those having a more complementary relationship to the base sequence of the complementary chain. Chains can be included.

The antisense polynucleotide is usually composed of about 10 to 1000 bases, preferably about 15 to 500 bases, and more preferably about 16 to 30 bases. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues (phosphates) of each nucleotide constituting antisense DNA are chemically modified with, for example, phosphorothioate, methylphosphonate, phosphorodithionate, etc. It may be substituted with a phosphate residue. These antisense polynucleotides can be produced using a known DNA synthesizer or the like.

Such antisense polynucleotides can hybridize to the RNA of the EC AT4 gene and can inhibit the synthesis or function of the RNA, or can interact with the RNA via the RNA. It can regulate and control the expression of AT4 gene.

The antisense polynucleotide of the present invention is useful for regulating and controlling the expression of ECAT4 in vivo and in vitro, and is useful for reliably eliminating undifferentiated cells as described above. is there. 5 'end hairpin loop of protein gene, 5' end 6—base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' The terminal palindrome region or the 3 ′ terminal hairpin loop can be selected as a preferable target region, but any region in the protein gene can be selected as the target. Regarding the relationship between the target nucleic acid and a polynucleotide complementary to at least a part of the target region, if the target nucleic acid can hybridize with the target region, the target nucleic acid is It means that it is “antisense”. Antisense polynucleotides can be polynucleotides containing D-reports, polynucleotides containing D-reports, N-glycosides of purine or pyrimidine bases, or other types of polynucleotides. Other polymers having a nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special bonds (however, such polymers are found in DNA and RNA) Such as nucleotides having a configuration that allows base pairing and base attachment). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, and may further comprise unmodified polynucleotides (or unmodified oligonucleotides). ), With known modifications, e.g., labeled, capped, methylated, or substituted with one or more natural nucleotides, as known in the art. Modified with an intramolecular nucleotide, for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramide, oleambamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate) , Such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Compounds having side-chain groups such as guanyl peptides, poly-L-lysine, etc., sugars (eg, monosaccharides), compounds having interactive compounds (eg, acridine, psoralen, etc.), chelating compounds ( For example, those containing metals, radioactive metals, boron, oxidizing metals, etc., those containing alkylating agents, and those with modified bonds (eg, alpha-anomeric nucleic acids, etc.). May be. Here, “nucleoside”, “nucleotide”, and “nucleic acid” may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with octalogens and aliphatic groups, or functional groups such as ethers, amines, etc. May be converted to The antisense polynucleotide of the present invention may comprise RNA, DNA or modified nucleic acid (RNA , DNA). Specific examples of the modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, a polynucleoside amide / a nucleic acid that is resistant to degradation of oligonucleoside amide, and the like.

The antisense polynucleotide of the present invention can be designed, for example, as follows. In other words, it makes the antisense polynucleotide more stable in the cell, enhances the cell permeability of the antisense polynucleotide, increases the affinity for the target sense strand, and In some cases, the toxicity of the antisense polynucleotide is reduced. Many such modifications have been reported, for example, in Pharm Tech Japan, vol. 8, p. 247 or p. 395, 1992, Antisense Research and Appli- cations, CRC Press, 1993.

The antisense polynucleotide of the present invention may be provided in a special form such as ribosome or microsphere, or may be provided in an added form more suitable for gene therapy. Examples of such additional forms include polycations, such as polylysine, which act to neutralize the charge of the phosphate backbone, and lipids, which increase the interaction with cell membranes and increase the uptake of nucleic acids. (Eg, phospholipids, cholesterol, etc.). Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.). These can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, or intramolecular nucleoside linkage. Other groups include capping groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonucleases and RNases. No. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of an antisense polynucleotide can be examined using the screening method of the present invention.

As described above, the antisense polynucleotide of the present invention may be double-stranded, and binds to RNA encoding ECAT4 and destroys the RNA or suppresses its function. That is, a part of the RNA encoding ECAT4 and its complementary Double-stranded RNAs containing various RNAs are also included in the antisense polynucleotide of the present invention.

In more detail, the use of the antisense polynucleotide is similar to that of ordinary gene therapy. For example, an antisense oligonucleotide or a chemically modified product thereof is directly administered to a target cultured cell or tissue. This can be performed by a method for controlling the expression of the target gene. The antisense polynucleotide of the present invention can be administered to cells as it is or by incorporating it into a vector for gene therapy. Also in these cases, the dose and the administration method vary depending on the type of cells, the number of cells, and the like, and those skilled in the art can appropriately select them.

The antisense polynucleotide or a chemically modified product thereof can bind to the sense strand mRNA in cells and regulate the expression of the target gene, that is, the expression of ECAT4, and thus the function (activity) of ECAT4. Can be controlled.

In the method of directly administering an antisense polynucleotide or a chemically modified product thereof to cultured cells, the antisense polynucleotide or the chemically modified product used is preferably 10 to 1000 bases, more preferably 15 to 500 bases, and most preferably Preferably, it should have a length of 16 to 30 bases. Upon administration, the antisense oligonucleotide or a chemically modified product thereof can be formulated (reagentized) using a commonly used stabilizer, buffer, solvent or the like.

The method using the above-mentioned recombinant virus includes, for example, the virus genome of the present invention such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus, vaccinia virus, polio virus, and symbis virus. There is a method of incorporating an antisense polynucleotide into a living body and introducing it into a living body. Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus or the like is particularly preferable. Examples of the non-viral introduction method include a ribosome method and a lipofectin method, and a liposome method is particularly preferable. Other non-viral introduction methods include, for example, a microinjection method, a calcium phosphate method, and an electroporation method.

The cell differentiation promoting agent of the present invention includes the above-mentioned antisense polynucleotide or a chemically modified product thereof, a recombinant virus containing the same, and an infection into which these viruses have been introduced. A cell or the like is used as an active ingredient. The agent for promoting cell differentiation of the present invention can be used as a reagent or a medicine. The administration form and the like of the composition can be appropriately determined depending on the target cell or tissue. The medicament or reagent of the present invention may be, for example, a form in which a virus vector containing the antisense polynucleotide of the present invention is embedded in ribosomes or membrane fusion liposomes (eg, Sendai virus (HVJ) -ribosome). it can. These ribosome formulations include suspensions, cryogens, and centrifuged concentrated cryogens. Further, the vector containing the antisense polynucleotide of the present invention may be in the form of a cell culture solution infected with the introduced virus. The dosage of the active ingredient in these various forms of preparation can be appropriately adjusted depending on the purpose. Usually, in the case of an antisense polynucleotide against the ECAT4 gene, about 0.001 x -lOOmg, preferably about 0.001 g-lOmg may be added / administered once every several days to several months. For example, in the case of a 10 cm Petri dish, 0.0001 ag to 1 OOfflg, preferably 0.001 ag to 1 Omg of the antisense nucleotide of the present invention may be added once every few days to several months.

Furthermore, the present invention provides (i) a double-stranded RNA containing a part of the RNA encoding ECAT4 and its complementary RNA, (ii) a medicine containing the double-stranded RNA, (iii) an EC A lipozyme containing a part of RNA encoding AT4, (iv) a medicine containing the lipozyme, (V) an expression vector containing a gene (DNA) encoding the lipozyme, and the like are also provided. Similarly to the above-mentioned antisense polynucleotide, double-stranded RNA, lipozyme and the like can also break down RNAs transcribed from the DNA of the present invention or inhibit its function. It can be used as a reagent or therapeutic agent such as double-stranded RNA, lipozyme which can suppress the function of ECAT4 or the DNA encoding it.

Double-stranded RNA can be designed and manufactured based on a polynucleotide sequence encoding ECAT4 according to a known method (eg, Nature, 411, 494, 2001). The lipozyme can be designed and manufactured based on the sequence of the polynucleotide encoding ECAT4 according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by substituting a part of a known lipozyme sequence with a part of RNA encoding ECAT4. ECA As a part of the RNA encoding T4, a sequence near a consensus sequence NUX (where N indicates all bases and X indicates a base other than G) which can be cleaved by a known lipozyme, and the like can be mentioned. Can be When the above-mentioned double-stranded RNA or lipozyme is used as the above-mentioned reagent or medicine, it can be prepared and administered in the same manner as the antisense polynucleotide. In addition, the above-mentioned expression vector is used in the same manner as a known gene therapy method and the like, and is used as the above reagent or medicine.

(3) ECAT4 enhancer, ECAT4 promoter region, consensus sequence recognized by ECAT4, recombinant vector and transformed cell of the present invention

The “ECAT4 enhancer” of the present invention refers to a base sequence that promotes the transcription of ECAT4. Use of the ECAT4 promoter region including the ECAT4 enhancer of the present invention enables efficient transcription of the ECAT4 gene and the like.

The ECAT4 enhancer of the present invention comprises (i) an ECAT4 enhancer comprising a part or all of the nucleotide sequence represented by SEQ ID NO: 7, (ii) SEQ ID NO: 17, SEQ ID NO: 18 or a sequence thereof. An EC AT4 gene containing the nucleotide sequence represented by SEQ ID NO: 19, (iii) a part of the nucleotide sequence represented by SEQ ID NO: 15, and represented by SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 ECAT4 enhancer containing the nucleotide sequence (iv) ECAT4 enhancer containing the nucleotide sequence represented by SEQ ID NO: 15, (V) a part of the nucleotide sequence represented by SEQ ID NO: 14, (Vi) one or more bases are deleted or replaced in the base sequence of the ECAT4 enhancer according to any one of the above (i) to (V). ECAT 4 (Vii) a DNA comprising the nucleotide sequence of ECAT4 enhancer described in any one of the above (to (v) and a stringent, which has the ability to promote transcription of offspring. An EC AT4 enhancer characterized in that it hybridizes under various conditions and has the ability to promote the transcription of the ECAT4 gene.

As a specific example of (i), for example, the DN of the region from position -4737 to position -4611 of the nucleotide sequence shown in FIG. 8 (Β) (the region from position 1 to position 127 in SEQ ID NO: 15) ECAT4 enhancer containing ン, and the region from position -4497 to position -4387 of the nucleotide sequence shown in FIG. 8 (B) (the region from position 241 to position 351 in SEQ ID NO: 15) ) DNA-containing EC AT4 enhancer.

In the present invention, the “promoter region” is a type of regulatory gene and is a region on DNA that determines the transcription initiation site of a gene and directly regulates its frequency. To induce promoter elements under cell-type or tissue-specific regulation, or promoter-dependent gene expression induced by exogenous signals or factors (eg, transcriptionally activated proteins) It may contain sufficient promotion elements.

The “ECAT4 promoter region” of the present invention contains the aforementioned ECAT4 enhancer of the present invention, and may be any DNA as long as it is a DNA having the promoter activity of ECAT4. . Specifically, for example, DNA consisting of a part or all of the base sequence shown in SEQ ID NO: 14 and the like can be mentioned.

The “consensus sequence recognized by ECAT4” of the present invention is a gene sequence of a site recognized by ECAT4 when ECAT4 binds to a gene and exhibits a transcription control action.

The consensus sequence recognized by ECAT4 of the present invention includes:

(i) a consensus sequence recognized by ECAT4, comprising a DNA described in any of (a) to (c) below:

(a) a DNA comprising a sequence containing the base sequence represented by SEQ ID NO: 4,

(b) a DNA consisting of a base sequence in which one or more bases are deleted, substituted or added in the base sequence represented by SEQ ID NO: 4, and which has an ability to bind to ECAT4;

(c) a DNA which hybridizes with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 4 under stringent conditions, and has an ability to bind to ECAT4,

(ii) a consensus sequence recognized by ECAT4, comprising a DNA described in any of (a) to (c) below:

(a) a DNA comprising the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 16,

(b) a base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16 wherein one or more bases are deleted, substituted or added, and DNA capable of binding CAT4,

(c) a DNA having the ability to hybridize with DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16 under stringent end conditions, and to bind to ECAT4;

(iii) a consensus sequence recognized by ECAT4, which comprises a part of the sequence described in (0 or (ii),

Can be mentioned. Here, “one or more bases” is preferably one to three bases, and more preferably one to two bases. The “part of the sequence” in (iii) may be, for example, the DNA of 5 to 10 nucleotides from the side end of the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16, or 3 'DNA from several bases to 10 bases from the side end can be mentioned, but is not limited thereto.

The ECAT4 enhancer of the present invention, the consensus sequence recognized by ECAT4, and the ECAT4 promoter region are human or other mammalian cells (for example, human lymphocyte cells, mouse fibroblasts, mouse ES cells, It may be any of genomic DNA, cDNA and synthetic DNA derived from rat ES cells, human ES cells) or any tissue where such cells exist.

Examples of the method for preparing the ECAT4 enhancer, the consensus sequence recognized by ECAT4, and the ECAT4 promoter region of the present invention include chemical synthesis, PCR, and hybridization.

When preparing using a chemical synthesis method, an automatic DNA synthesizer, for example, a DNA synthesizer model 38OA (manufactured by ABI) can be used.

Next, a method for preparing the ECAT4 enhancer or ECAT4 promoter overnight region of the present invention using PCR will be described. A genomic library of type とする is described, for example, in "Mocular C 1 oning: AL abo rato ry Manua 1 2nd edition" (1989), Cold Spring Harbor Labo ratory Press, etc. It can be prepared from tissues of mammals such as humans and mice according to the methods described. In addition, commercially available genomic DNA such as human genomic DNA (manufactured by Clonetech) and commercially available genomic libraries such as human genome walker kit (manufactured by Clonetech) can be used. Next Then, perform PCR using primers corresponding to the promoter to be amplified. The primer can be appropriately designed based on the nucleotide sequence represented by SEQ ID NO: 7, SEQ ID NO: 14 or SEQ ID NO: 15, and a restriction enzyme recognition sequence or the like is added to the 5 ′ end thereof. Is also good. The DNA amplified as described above is described in `` Mo ecu 1 ar C 1 oning: AL abo rato ry Manu al 2nd edition '' (1989), Cold Spring Har Harbor rboratory Press, Cu rr ent Protocols In Mo 1 ecu 1 ar Biol ogy ”(1987), John Wiley & Sons, Inc. ISBN 0—471—50338—X It can be cloned into a vector according to the method. Specifically, for example, cloning can be performed using a plasmid vector included in a TA cloning kit manufactured by Invitrogen or a plasmid vector such as pB1uescript II manufactured by Stratagene. The nucleotide sequence of the cloned DNA is described in F. Sanger, S. Nickl en, AR Cou ls on, Proceedings of Na ti on al Ac ad emy of S ci ence USA (1977), 74, 5463_ The analysis can be carried out by the didoxita-mining method described in 5467 and the like.

Next, a method of preparing using a hybridization method will be described. First, the DNA used for the probe is labeled. The DNA used for the probe may be a DNA having at least a part of a base sequence such as an enhancer to be prepared or a promoter region, for example, a base sequence represented by any one of SEQ ID NOs: 4 to 7 and 14 to 19 or DNA comprising a continuous partial base sequence and having a chain length of 16 bases or more and 160 bases or less, wherein one or more bases are deleted, substituted or added in the base sequence of the DNA And DNA that hybridizes with the DNA under stringent conditions.

The DNA used for the probe can be obtained, for example, by the above-mentioned usual DNA preparation method such as a chemical synthesis method, a PCR, and a hybridization method. In addition, the DNA used for the probe is a gene that encodes ECAT4 by itself. It may have the ability to control the photo.

In order to label the DNA used for the probe with a radioisotope, for example, Random Lab elling Kit manufactured by Ba Ringer and Takara Shuzo can be used, and dCTP in the ordinary PCR reaction composition can be used as [a - ³² P] instead of dCTP, by performing to PC R reacting the DN a to鍀型used to probe, it is also possible to perform labeling. When the DNA used for the probe is labeled with a fluorescent dye, for example, ECL Direct Nucleic Acid Label and D itection on Syst em mersh am Pharmacia Biotech) can be used. . As a DNA library for hybridizing probes, for example, a genomic DNA library derived from animals such as rodents such as mice can be used. As the DNA library, a commercially available genomic DNA library can be used, and “Moecular C 1 oning: AL aboratory Manu al 2nd edition” (1989), Cold Spring Harr Borr Laboratory Press, "CurrentProtocols In Molecular BiologyJ (1987), John Wiley & Sons, Inc. IS BN 0—471—50338_X, etc. According to the usual method for preparing a library described in (1), for example, using a λ vector such as λ FIXII, λ EMBL 3, λ EMBL4, λ DASH II manufactured by Stratage, Gig ap ackpack A genomic DNA library can be prepared using in vitro packaging (Stratagene) or the like and used.

Examples of the hybridization method include colony hybridization and plaque hybridization, and it is preferable to select a method according to the type of the vector used for preparing the library. For example, if the library used is a library constructed with a plasmid vector, colony hybridization can be performed. Specifically, first, transformants are obtained by introducing the library DNA into a host microorganism, and the obtained transformants are diluted, plated on an agar medium, and cultured at 37 until colonies appear. Also used la If the library is a library constructed with a phage vector, black hybridization can be performed. Specifically, first, the host microorganism and the phage of the library are mixed under conditions that allow infection, and then mixed with a soft agar medium, which is then spread on an agar medium. Incubate at 37 ° C until plaques appear. More specifically, for example, Mo 1 ecu 1 ar Cl on i ng 2 nd edit on (J. Sam Rok, EF Frisch, T. Maniatis, Cold Spring Har bor L abo rato ry P ress 1989 years) 2. according to the method described in 2.65, etc. 60, a density of 0. 1 to 1. O pfu agar lmm ² per a NZY agar medium, about 9. 0 X 10 ⁵ pfu file spread one di libraries, cultured 6-10 hours at 37 ° C.

Next, in any of the above-mentioned hybridization methods, a membrane filter is placed on the surface of the agar medium on which the above-mentioned culture has been carried out, and the transformed cells or phage carrying the plasmid are placed in the membrane. Transfer to a plain filter. This membrane filter is subjected to an alkali treatment, a neutralization treatment, and a treatment for fixing DNA to the filter. More specifically, for example, in the case of plaque hybridization, the cloning and sequence are carried out according to the usual method described in a manual for plant biotechnology experiments (edited by Watanabe and Sugiura, Rural Bunkasha 1989) and the like. Place a dinitrocellulose filter or a nylon filter, such as Hybond-Ν '(Amersham Pharmacia Biotech), on the agar medium and let it stand for about 1 minute to allow the phage particles to adsorb to the membrane filter. . Next, the filter was immersed in an alkaline solution (1.5 M sodium chloride, 0.5 N sodium hydroxide) for about 3 minutes to dissolve the phage particles and elute the phage DNA over the filter. Perform a treatment of immersion in a neutralization solution (1.5 M sodium chloride 0.5 M Tris-HCl, H7.5) for about 5 minutes. After washing the filter with a washing solution (300 mM sodium salt, 3 OmM sodium citrate, 200 ηιΜtris-hydrochloric acid) for about 5 minutes, for example, by baking at about 80 ° C for about 90 minutes, the phage is removed. Immobilize the DNA on the filter. Hybridization is performed using the filter thus prepared and the probe. Reagents and temperature conditions for performing hybridization are described in, for example, “DNA cl on in DMG 1 o Ver”. , Ar ac ticalapproach ”I RL PRESS (198 5) I SBN 0—947946—18—7, Cloning and Sequence: Plant Biotechnology Experiment Manual (edited by Watanabe, Sugiura, Rural Culture Co., 1989), or Mo lecular C l on i ng 2nd editi on (J. S amb ro ok, EF Frisch, T. Man iatis, Cold S pri ng Har bor lab ab oratory Press, 1989) Can be done. For example, it contains 450-90 OmM sodium chloride, 45-9 OmM sodium citrate, contains sodium dodecyl sulfate (SDS) at a concentration of 0.1-1.0% (W / V), Prehybridization containing specific DNA at a concentration of 0 to 200 gZmL and optionally containing albumin, ficoll, polyvinylpyrrolidone, etc. at a concentration of 0 to 0.2% (W / V). Chillon solution, preferably prehybridization containing 90 OmM sodium chloride, 9 OmM sodium citrate, 1% (W / V) SDS and 100 gZmL denatured Ca1f-thymus DNA the Zeshiyon solution, prepared in the ratio of the filter one 1 cm ² per 50 to 200 were prepared as L, 1 to 4 hours at 42 to 68 ° C by immersing the filter one to the solution, preferably Incubate at 45 ° C for 2 hours. Then, for example, containing 450-90 OmM sodium chloride, 45-9 OmM sodium citrate, containing SDS at a concentration of 0.1-1.0% (W / V), denatured non-specific A hybridization solution containing DNA at a concentration of 0 to 200 g / mL and optionally containing albumin, ficoll, polyvinylpyrrolidone, etc. at a concentration of 0 to 0.2% (WV), Preferably, a hybridization solution comprising 90 OmM sodium chloride, 9 OmM sodium citrate, 1% (W./V) SDS and 100. ". GZmL of denatured Calf-thymus DNA, methods in the preparation obtained was pro one Bed (filter 1 cm ² per ^{1. 0X 10 4 ~2. 0 X} 10 6 c pm equivalent) and the solution from 50 to 01 ² per Ji filter 1 which combined mixed with Prepare the solution at a rate of 200 L, immerse the fill in the solution, and keep at 42-68 ° C for 4-20 hours, preferably at 45 ° C for 16 hours. After the hybridization reaction, remove the filter, and remove 15-30 OmM sodium chloride, 1.5-30 mM. Sodium citrate, and a washing solution at 42 to 68 ° C. containing 0.1 to 1.0% (W / V) SDS, etc., preferably 30 OmM sodium chloride, 3 OmM citrate Perform 1 to 4 filter washes for 10 to 60 minutes with a wash solution containing sodium and 1% (W / V) SDS at 55 ° C, preferably 2 washes for 15 minutes. Furthermore, the filter is lightly rinsed with 2XSSC solution (containing 30 OmM sodium chloride and 3 OmM sodium citrate) and then dried. This filter is subjected to, for example, autoradiography to detect the position of the probe on the filter, thereby detecting the position of the DNA that hybridizes with the used probe on the filter. By identifying a clone corresponding to the position of the detected DNA on the filter on the original agar medium and picking it, a clone having the DNA can be isolated. Specifically, for example, the filter is exposed to an imaging plate (Fuji Film) for 4 hours, and then the plate is analyzed using BAS 2000 (Fuji Film) to detect a signal. A portion of the agar medium used for the preparation of the film, which corresponds to the position where the signal was detected, was cut out into a square of about 5 mm, and this was cut into about 500 L of SM buffer (5 OmM Tris-HCl pH 7.5, Elute phage particles by soaking in 0.1M sodium chloride, 7mM magnesium sulfate, and 0.01% (W / V) gelatin for 2-16 hours, preferably 3 hours. The obtained phage particle eluate was subjected to Molecular Cloning 2ndedition (J. Sam Rook, EF Frisch, T. Maniatis, Cold Sp rig Har Har bo r Labo rato ry

Press, 1989) 2. Spread on an agar medium according to the method described in 60 to 2.65, and culture at 37 for 6 to 10 hours. Using this agar medium, phage DNA is immobilized on the filter in the same manner as described above, and hybridization is performed using this filter and the above-mentioned probe. Phage particles were eluted from the portion of the agar medium used to prepare the filter, which corresponded to the position where the signal was detected, spread on the agar medium, and a filter was prepared in the same manner as described above, and hybridized. Perform the dimensioning. By repeatedly specifying and purifying such a phage clone, a phage clone containing a DNA consisting of a base sequence that hybridizes with the probe used can be obtained. Screening by hybridization as described above The DNA contained in the clone obtained as described above is subcloned into a plasmid vector, which is easy to prepare and analyze, such as commercially available pUC18, pUC19, pBLUESCR I PT KS +, pBLUESCR I PT KS-, etc. DNA was prepared and prepared by F. Sanger, S. Nickl en, AR Cou 1 s on, Proceeding ng sof Na ti al al Academy of Sci ence USA (1977), 74, 5463—5467. The nucleotide sequence can be determined using the didoxinating method described in, for example. The preparation of the sample used for the nucleotide sequence analysis is performed, for example, by Molecular Cloning 2nd edition (J. Sam Rook, EF Frisch, T. Maniatis, Cold Spring Harbor L aboratory Press, 1989) 13. It can be carried out according to the primer extension method described in 15 and the like. In addition, the phage clone was prepared using the Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Laboratory Press, 1989). 2.Amplify in NZYM liquid medium according to the method described in 2.60 to 2.65, etc., and prepare a phage solution.From this, for example, Lambda_TRAPPLUS DNA Isolation Kit (C1ontech) The phage clone DNA can be extracted by using, etc., and the DNA can be used as a type II, for example, by preparing a sample for base sequence analysis by the above-mentioned primer extension method and analyzing the base sequence. The ability of the DNA thus obtained to control the transcription of the ECAT4 gene can also be confirmed as described below. In the present application, “the ability to control transcription” means, for example, an activity of initiating and / or promoting transcription of a gene located downstream of a promoter region (hereinafter, also referred to as promoter activity). ).

The ECAT4 enhancer of the present invention, the consensus sequence recognized by ECAT4, and the ECAT4 promoter region have mutations in the sequence containing the base sequence shown in any one of SEQ ID NOs: 4 to 7 and 14 to 19. It may be made by introducing. Specifically, for example, the method described in A. Greener, M. Callahan, Strategies (1994) 7, 32-34, etc. is used at random. Can be obtained by introducing a mutation, such as W. Kramer, eta 1., Nucleic Acids Research (1984) 12, 9441, or W. Kramer, HJ Frits, Method sin En. z ymo l ogy (1987) 154, 350, etc., gapped duplex method (g app eddup 1 ex) method, or TA Knoke Proc. of Natl. Ac ad. Sci. USA (1985) 82, 488 or TA Kun ke 1, eta 1., Method sin Enzymolgy (1987) 154, 367, etc. The site-specific mutation is introduced using the Kunke 1 method. Can be obtained by: Alternatively, a chimeric DNA is prepared in which one or several partial nucleotide sequences of the EC AT4 promoter region including the nucleotide sequence shown in SEQ ID NO: 15 are replaced with a part of the DNA of another promoter. For example, S. Hen i ko ff, eta 1., Gene (1984) 28, 351, C. Yan isch—Pe rr on, eta 1., Gene (1985) ) The method described in 33, 103 etc. can be used.

The promoter region of the present invention prepared by the above-described various methods can be prepared by a conventional method, for example, “Tamura Takaaki (published by Yodosha), New Transcription Control Mechanism (2000)”, pp. 33-40, “Nomura According to the method described in Shintaro and Toshiki Watanabe supervised by Shujunsha (published by Shujunsha Publishing Co., Ltd., Deisotope Experiment Protocol (1998)), a part of the base is deleted while maintaining the promoter activity. (That is, prepared by excising with an appropriate restriction enzyme) can also be used as the promoter region of the present invention. The ability of the obtained DNA to regulate the transcription of the ECAT4 gene can be confirmed by the method described below.

In preparing the recombinant vector of the present invention, the ECAT 4 enhancer or ECAT 4 promoter region of the present invention (hereinafter, referred to as “E CAT 4 enhancer / promo overnight region” in the present specification) is inserted. The recombination vector may be any recombination vector that can function in a desired cell. When using the EC AT enhancer of the present invention, a known suitable promoter can be operably linked. The recombinant vector of the present invention selects cells into which the recombinant vector has been introduced. For example, a kanamycin resistance gene, an octaidaromycin resistance gene, a neomycin resistance gene, and the like. Further, in the recombination vector, a position where the enhancer-promoter region of the present invention and a desired gene are operably linked on a recombinant vector, for example, the enhancer-Z promoter region. When a gene insertion site is further provided downstream of the site into which the gene is inserted, it can be preferably used for construction of a recombinant vector for expressing a desired gene in cells. Here, the gene insertion site is, for example, a nucleotide sequence that can be specifically recognized and cleaved by a restriction enzyme usually used in genetic engineering techniques, and is present only on a recombinant vector having a region of the enhancer / promoter of the present invention. Preferred types of restriction enzyme recognition sequences are preferred. Specific examples of the recombinant vector include a pUC plasmid (pUC118, pUC119 (manufactured by Takara Shuzo) and the like), a pSC101 plasmid, and a pBR322 plasmid (manufactured by Boehringer Mannheim). Can be In the preparation of the recombinant vector of the present invention, the recombinant vector for inserting the consensus sequence recognized by the ECAT 4 of the present invention may be any recombinant vector that can function in a desired cell. It may also contain a marker gene for selecting cells into which the recombinant vector has been introduced (for example, a kanamycin resistance gene, a hygromycin resistance gene, a neomycin resistance gene, etc.). In the recombination vector, a position where the consensus sequence of the present invention and a desired gene are operably linked on the recombination vector, for example, downstream of a site where the consensus sequence is inserted. If a suitable promoter and gene insertion site are further provided, it can be preferably used for construction of a recombinant vector for expressing a desired gene in cells. Here, the gene insertion site is, for example, a nucleotide sequence that can be specifically recognized and cleaved by a restriction enzyme usually used in genetic engineering techniques, and is a recombinant having a consensus sequence recognized by ECAT4 of the present invention. Restriction enzyme recognition sequences of the type present solely on the vector are preferred. Specific examples of the recombinant vector include a pUC plasmid (pUC118, pUC119 (Takara Shuzo) etc.), a pSC101 plasmid, and a pBR322 plasmid (Boehringer Mannheim). Be mentioned

In addition, E CAT 4 enhancer / promoter. In order to examine the binding of the consensus sequence recognized by ECAT4, a detectable structural gene may be ligated downstream of the ECAT4 enhancer / promoter region or the consensus sequence recognized by ECAT4. Various repo overnight genes are used as structural genes linked downstream of the enhancer / promoter region or consensus sequence. As the repo overnight gene, luciferase gene, CAT (Chloramphenicol acetyl transferase) gene, alkaline phosphatase gene, and] -galactosidase gene are widely used. Any other structural gene can be used as long as there is a method for detecting its gene product.The above-mentioned structural gene can be incorporated into a vector by recognizing the ECAT4 enhancer / promoter region or ECAT4. The above-mentioned structural gene may be ligated to an appropriate restriction enzyme cleavage site located downstream of the consensus sequence, for example, a commercially available vector such as pGL3 Basic, pGL3 promoter (Promega). It can also be used.

As a host transformed with the above-described recombinant vector, for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, animal cells, and the like are used. Examples of animal cells include monkey cell COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CH O (hereinafter CH〇 (dhfr_) cell) ), Mouse L cells, mouse AtT—20, mouse myeloid cells, rat GH3, mouse fibroblasts 3T3—L1, human liver cancer cells HepG2 (hereinafter referred to as HepG2 cells). Abbreviations), human osteosarcoma cells MG-63 (hereinafter abbreviated as MG-63 cells), human FL cells, white adipocytes, egg cells, ES cells (Evans, MJ and Kaufman, Η. (1981) Nature, 292, 154). Preferred are mammalian stem cells or ES cells derived from mammals, and particularly preferred are mouse ES cells, rat ES cells and human ES cells.

Methods for transforming these cells include the calcium phosphate method (Graham et al. (1973) Virology, 52,456 Elect-mouth poration method (Ishizaki et al. (1986) Cell Engineering, 5, 577), microinjection method, lipid for gene transfer. (Lipofectamine, Lipofectin; Gibco-BRL), etc. More specifically, To transform animal cells, see, for example, Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). It can be performed according to the method described. The transformant is cultured in the presence of a specific compound, and the ability to control the promoter activity of the compound can be known by measuring and comparing the amount of the gene product in the culture. The transformant is cultured by a method known per se. The pH of the medium is preferably adjusted to about 5-8.

If the recombinant vector of the present invention is a DNA containing the ECAT4 enhancer or the promoter region of ECAT4, the above transformant can be used to obtain the ECAT4 enhancer or ECAT4 promoter. Compounds that regulate (activate / suppress) the activity of the region can be screened. In addition, if the recombinant vector of the present invention is a DNA containing a consensus sequence recognized by ECAT4, the use of the above transformant controls the binding of ECAT4 to the consensus sequence (activation Z suppression). ) Can be used for screening compounds.

(4) A method for screening a substance that regulates (activates / suppresses) the activity of the ECAT4 enhancer or ECAT4 promoter region, and a screening method using a consensus sequence recognized by ECAT4.

A substance that controls (activates / suppresses) the expression of ECAT4 by using the ECAT4 gene, ECAT4 enhancer, ECAT4 promoter region, or a consensus sequence recognized by ECAT4 of the present invention, or It can screen substances that control the function of ECAT4 (suppress activation Z).

For example, a substance that activates or suppresses ECAT4 expression can be obtained by introducing a chimeric gene in which a luciferase gene is linked to the base sequence of the ECAT4 enhancer / promoter region into cells (for example, ES cells). You can screen. Similarly, by introducing a chimeric gene of a promoter gene containing a consensus sequence recognized by ECAT4 and a repo overnight gene into cells (for example, ES cells, etc.), transcriptional activation or transcription by ECAT4 is performed. Substances that affect suppression or have the same activity as ECAT4 can be screened. In addition, a well-known Binding Attach, for example, SPA method (Amersham Armasia) It is also possible to search for a substance that inhibits the binding between ECAT4 and the consensus sequence of the present invention using the binding assay or the like used.

The method of screening for a substance that controls the activity of the ECAT4 enhancer or ECAT4 promoter region of the present invention includes the following steps (a), (b) and (c):

(a) contacting a test substance with a cell transformed with DNA in which an ECAT4 enhancer or an ECAT4 promoter region is linked to a repo overnight gene;

(b) measuring the activity of the reporter gene in the cells contacted with the test substance, and comparing the expression level with the activity in control cells not contacted with the test substance;

(c) a step of selecting a test substance that controls the activity of the ECAT4 enhancer or ECAT4 promoter region based on the comparison result of (b) above.

As the cells used for such screening, the transformant of the present invention described in the above (3) is useful.

The test substance (candidate substance) to be screened by the screening method of the present invention is not limited, but may be a nucleic acid (including the antisense nucleotide of the present invention), a peptide, a protein, an organic compound, an inorganic compound, or the like. Yes, the screening of the present invention is specifically carried out by bringing these test substances or a sample containing them (test sample) into contact with the cells. Examples of such test samples include, but are not limited to, a cell extract containing a test substance, an expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, a natural compound, and the like.

In the screening of the present invention, the conditions under which the test substance is brought into contact with the cells are not particularly limited, but it is preferable to select culture conditions (temperature, pH, medium composition, etc.) that do not kill the cells.

As shown in the Examples, cells in which ECAT4 was forcibly expressed maintain the totipotency of ES cells even in the absence of LIF and Fada-single cells, and are therefore master genes involved in the self-renewal ability of ES cells. It is believed that there is. Therefore, the screening method of the present invention includes a method of searching for a substance that controls the activity (suppression of activation Z) using the transcription ability of the ECAT4 enhancer promoter region as an index. By this screening method, candidate substances or ES cells that are effective for maintaining ES cells in culture can be identified. Candidate substances effective for differentiation can be provided. It is considered that a candidate substance that activates the transcription ability of the ECAT4 enhancer region is useful for maintaining ES cells in culture, and a suppressor substance is useful for differentiating ES cells.

More specifically, the activity of the ECAT4 enhancer / promoter region in cells to which the test substance (candidate substance) is added fluctuates (increases) compared to the level of cells in which the test substance (candidate substance) is not added. / Lower) can be selected.

As described above, the activity of the EC AT4 enhancer / promoter region is determined by connecting a marker gene such as a luciferase gene to a gene region (expression control region) that regulates the expression of the ECAT4 gene. It can be carried out by measuring the activity of a protein derived from the marker gene using a cell line into which the fusion gene has been introduced. Examples of the measuring method include, for example, measuring luciferase activity by a method according to the description of Brasier, A.R., et al. (1989) Biotecniques vol.7, 1116-1122.

The marker gene is preferably a structural gene of an enzyme that catalyzes a luminescence reaction or a color reaction. Specifically, as described in the above (3), in addition to the luciferase gene, a secreted alkaline phosphatase gene, a chloramphenicol-acetyltransferase gene, a β-dalc-mouth nidase gene, Reporter genes such as the galactosidase gene and the aequorin gene can be exemplified. Example

Hereinafter, the present invention will be described specifically with reference to examples. However, the present invention is not limited to these examples, and it goes without saying that ordinary changes in the technical field of the present invention can be made.

Example 1

ECAT4 identification

In order to identify major transcription factor candidates of pluripotent cells, a digital differential display developed by Nation a 1 Center for Biologic Informaton was performed here. This method enables expression sequence tag (EST) libraries derived from various cells and tissues to be stored in libraries. It is possible to compare the expression frequency of each gene in the present invention. This time, we compared an EST library derived from mouse ES cells (clone 27705) and an EST library derived from various somatic tissues and organs (clone 1328835).

(1) Digital differential display

In the present invention, using the Digital D ifferential D isplay program (http: // www, ncbi.nlm.nih.gov/Unigene/ddd/cgi), it is possible to obtain c cells derived from ES cells and other tissues. Gene expression in the DNA library was analyzed. The ES cell-derived EST libraries were # 274 and 220 (total 27705 entries). The libraries used as representatives of various somatic cell tissues are # 467, 318, 198, 408, 239, 400, 453, 109, 16, 523, 161, 483, 258, 264, 419, 529, 327, 41 1, 417, 418, 399, 196, 271, 255, 495, 101, 98, 351, 416, 321, 251, 412, 379, 549, 329, 265, 449, 328, 516, 320, 436, 427 , 297, 366, 390, 315, 228, 277, 292, 284, 285 and 30 (total of 1328835 entries).

As a result of analysis by the program, 20 genes were found at least 20 times higher in ES cells than in somatic tissues. These include experimentally identified ES cell-specific marker genes such as Oct 3/4, UTF 1 (Okud aeta 1., 1998), and Re 1 (Roerseta 1., 1991). Exist, demonstrating the usefulness of this technology. Northern blot analysis confirmed ES cell-specific expression of at least nine genes whose characteristics were not known.Here, one of them, which has a homeobox domain, is called ECAT4. (Fig. 1 (A)).

Transcription of £ 〇haccho 4 was observed in undifferentiated 18 (1 \ [6 1116 eta 1., 1996) and MG 1.19 (Gs sma nneta 1., 1995) ES cells (Fig. )), Decreased by differentiation induced by retinoic acid. No ECAT4 transcript was detectable in 12 adult mouse organs. Further anti-E CAT 4 polyclonal antibodies were generated, and the four ESs were £ 8, 8, MG1.19, J1 (Lieta 1., 1992), and CGR 8 (Nicholseta 1., 1 990). Expression was confirmed in the cell line (Fig. 1 (C)). Treatment with retinoic acid suppressed the expression of EC A T4 in all four lines. These data indicate that the specific expression of a new homeobox gene, ECAT4, is a property commonly found in ES cells.

Example 2

E CAT 4 protein and a new home box

The 2184 base full-length cDNA of ECAT4 was isolated by the 5 'RACE method (Fig. 1 (A)). And a single gene encoding a 305 amino acid polypeptide.

—Pun reading frame identified.

A homology search of this ECAT4 cDNA with a relatively long 377 untranslated region of 1077 bases revealed that the ECAT4 protein contains a homeobox domain. According to phylogenetic analysis, this ECAT4 home box is Nk

-2 gene family (Harveye, 1996) (Fig. 2 (B)). However, amino acid identity is less than 40%. Further EC A

In T4, valine is present at a position corresponding to tyrosine, which is strictly conserved in the NK-2 family. The TN-2 and NK-2 specific domains that are conserved in the NK-2 family are absent from ECAT4. From these data EC

It is suggested that AT4 is not a NK-2 family. ECAT4 is Oct 3

For homeobox domains in other homeobox genes expressed in ES cells, such as Z 4., Pern (Fan eta 1., 1999) and Ehox (Jacks on eta 1., 2002) Have low homology. There is no known domain of the EC AT4 protein other than the home box. Thus, ECAT4 is considered to be a new homebox transcription factor that does not belong to any of the known subfamilies.

Example 3

Target disruption of mouse ECAT4 gene in mouse ES cells (1) Target disruption of mouse ECAT4 gene

Exon 2 of the mouse ECAT4 gene having a homeobox domain was replaced with IRES (internal ribosome binding site) -β-geo (fusion of j3 galactosidase and neomycin resistance gene) Cassette (Mound tfordeta 1., 19994) Alternatively, a targeting vector for replacing the hygromycin resistance gene was prepared. A 4 kb fragment containing intron 1 and a 1.5 kbp fragment from exon 3 to exon 4 were amplified from the BAC clone and used as the 5 'and 3' homology regions of the targeting vector (Fig. 2 (A )). The obtained evening targeting vector was introduced into RF8 ES cells by electroporation (Meinereta 1., 1996). Screening of genomic DNA for homologous recombination from G418-resistant colonies was performed by Southern blot.

Using the β-geo target vector, 27 of the 96 colonies screened were found to be positive. When the hygr vector was used, 8 out of 27 cells were positive. Confirmation of homologous recombination was performed using a Southern plot (Figure 2 (B)).

In order to obtain homozygous ES cells, in this study the hygr vector was introduced into heterozygous mutant cells established using the / 3-geo vector. Cells were sorted on MEF cells using LIF using hygromycin. In 34 of the 83 screened, homologous recombination of the hygr vector was confirmed. Of these, 11 clones were found to be homozygous for the ECAT4 mutation, both by Southern blot (FIG. 2 (B)) and PCR (FIG. 2 (C)). Northern plot (Figure 2 (D)) analysis confirmed the lack of ECAT4 expression in homozygous cells. In the other 23 clones, the hygr vector replaced the] 3-geo cassette.

Next, we attempted to create homozygous cells using the opposite approach. That is, the iS-geo vector was introduced into the heterozygous mutant cells established by the hygromycin vector. Cells were sorted using G418 on SIF feeders expressing LIF. Of the 397 screened, 114 homologous recombination of the i3-geo vector was confirmed. Of these, six clones are homozygous. It turned out. ECAT4-deficient cells obtained by both combinations exhibited the behavior as shown in Examples 4 and 5 below.

(2) PCR screening for homologous recombination

Initial screening for homologous recombination was performed by PCR using ExTaQ polymerase. By specific amplification using the 6047—ins Sl. 2 and 6047—int AS 1 primers, a 3.2-kb band from the wild-type locus and a target locus from the / 3—ge0 vector An 8.5-kb band was obtained, and a 4.2-kb band was obtained from the target locus of the hygr vector.

(3) Southern blot analysis for screening of homologous recombination

Confirmation of homologous recombination was performed using Southern plot analysis. 5 'Southern plot For analysis, genomic DNA was digested with BamHI, separated on a 0.8% agarose gel, and transferred to a nylon membrane as previously described (Yaman ak ata 1.

2000; Yamanaketa 1., 1998). Hybridization using a 470 bp probe from the 5 'flanking region resulted in an 8.8 kb band from the wild-type locus, 11.4 kb from the target locus using the β geo target vector. A 9.8 kb band was generated from the target locus using the hygromycin vector. Digestion was performed using Sca1 for 3 'Southern plot analysis. Hybridization using a 1 kb probe from the 3 'flanking region resulted in an 11 kb band from the wild-type locus, and 1) a 15.4-kb band from the target locus using the 3 geo target vector. A 7.3 kb band was generated from the target locus using the hygromycin vector.

(4) Identification of mouse genotype

After identification of correctly targeted ES cell clones, the gene type of the mouse was determined using the three primers PCR. The first sense primer, 6047-S5, was designed based on exon 2 to amplify the wild-type loci. The second sense primer] 3—geoscreening 1 was designed based on the j8 geo cassette to amplify the targeted locus. The common antisense primer 6047-int AS3 was designed based on intron 2 to amplify both wild-type and targeted loci. PCR using these primers resulted in an 850 bp fragment from the wild-type locus. A 600 bp fragment was generated from the targeted locus. PCR was performed using ExTaq (Takara) according to the manufacturer's protocol. The PCR program included initial denaturation (94 ° (: 2 minutes), 35 cycles (94 ° C 30 seconds, 53 ° C 30 seconds, 68X: 1 minute) and final extension (68 ° C 7 minutes). Was.

Example 4

Differentiation of ECAT4 homozygous mutant ES cells into extraembryonic endoderm

The colonies of ECAT4 deficient cells at the end of drug selection were morphologically different from those of wild-type and heterozygous ES cells. The central cell appeared undifferentiated, while the surrounding cells were differentiated. When maintained on SNL feeder cells by the addition of recombinant LIF, almost all cells differentiated rapidly and spontaneously into giant round cells (Figure 3 (A)). When cultured in the absence of feeder cells, ECAT4-deficient cells became even flatter and showed a morphology similar to the parietal endoderm. The growth rate of ECAT4-deficient cells was greatly inhibited (Fig. 3 (B)).

According to Northern plot (Fig. 3 (C)) and RT-PCR (Fig. 3 (D)) analysis, ECAT4 cells showed GATA4, GATA6, H inf 1/3, H inf 4 «, Coup—tfl, Coup- Primitive endoderm transcription factors such as tf II, laminin B1, dis ablled homolog 2 (Dab 2), Sp arc, tissue plasminogen activator (tPA), thrompomodulin (TM), etc. Expresses parietal endoderm markers and visceral endoderm markers such as α-fetoprotein (AFP), bone morphogenetic protein 2 (BMP 2), India an hedgehog (I hh), and transthyretin (T tr) Was. However, the mesodermal marker T. trophoblastoma — force Cdx 2, the primitive ectodermal force fibroblast growth factor (Fg f) — 5, or the neuroectodermal marker islet — 1 (I s 1 1) did not increase.

To confirm that ECAT4-deficient cells had lost pluripotency, these cells were injected into blastocysts of C57BL / 6 mice. When the heterozygous mutant cells were injected, a plurality of chimeric mice to which ES cells greatly contributed, as judged from the color of the fur, were obtained. In contrast, chimera mice were not obtained when ECAT4 homozygous mutant cells were injected, confirming that the pluripotency of these cells was lost.

Example 5 Early embryonic lethality in ECAT 4-deficient mice

To examine the invivo function of ECAT4, ECAT4 heterozygous ES cells obtained using the j3-geo vector were injected into C57BLZ6 blastocysts. Germline transmission was obtained from three independent ES clones. ECAT4 heterozygous mice were obtained at the expected Mendelian ratio, overall appearance was normal and reproductive. When the embryo before implantation was stained with X-ga1, expression of; 8-geo was detected only in the inner cell mass of the blastocyst (FIG. 4 (A)). Heterozygous crosses did not produce any homozygous offspring, indicating embryonic lethality.

To determine when embryos would die, we analyzed embryos of heterozygous crosses at various stages. At 7.5 dpc and thereafter, there were no homozygous embryos. The empty decidua of approximately 1Z3 indicated that ECAT4-deficient embryos died shortly after implantation. In contrast, homozygous and heterozygous blastocysts were found to be present by Mendelian ratio. However, when cultured on gelatin-coated plates, the inner cell mass of ECAT4-deficient blastocysts did not proliferate (Fig. 4 (B)). These data indicate that ECAT4 is essential for self-renewal of primitive ectoderm (epiblast).

Example 6

Efficient establishment of ES cells overexpressing ECAT4

(1) Preparation of ES cells overexpressing ECAT4

Using the supertransfection system, the coding region of ECAT4 was introduced into the pCAG-IP vector to construct a gene vector. The transgene vector was introduced into MG1 • 19 ES cells using Lip efo ctami n 2000 (Invitrogen) according to the protocol for ES cells provided by the manufacturer. Specifically, MG1.19 cells were seeded in a 6-well plate (1.2 x 10 5 cells swell), cultured for 24 hours, and 8 g of the transgene was transformed with 41 Lipofect ami n 2000. After forming the complex, it was added to the medium. The next day, the cells were seeded on a 100 mm Petri dish and selected using 2/2 g / ml puromycin. Sorting continued throughout the experiment.

In this transgene vector, the target gene is located downstream of a single CAG promoter. And the bite-mycin resistance gene are continuously present, and mRNA containing the two genes is transcribed. There is an internal ribosome entry site between the two, from which the puromycin resistance gene is translated. Therefore, all the cells that have become puromycin resistant will also express the target gene. In fact, using this method, ECAT4 was introduced into MG1.19 cells, and puromycin was selected. As a result, 90% or more of the cells showed ECAT4 expression. The resulting CAG-EC AT4 cells express significantly more EC AT4 transcripts (Fig. 5 (A)) and proteins (Fig. 5 (B)) compared to control cells transfected with the parental vector. did.

(2) LIF-independent self-renewal of forcedly expressed ES cells

ECAT4 levels in control cells decreased after removal of LIF, but CAG—EC

It was rather increased in AT4 cells. In the absence of LIF, control cells gradually differentiated and flattened (Fig. 5 (C)), expression of Oct 3/4 was suppressed (Fig. 5 (A)), and the growth rate of the cells was reduced by LIF. It was reduced by the removal of (Fig. 5 (D)). On the other hand, CAG-ECAT4 cells remain undifferentiated even after LIF removal (Fig. 5 (C)), and expression of Oct 3/4 continues (Fig. 5 (A)). It also showed resistance to growth suppression caused by LIF removal (Fig. 5 (D)). These data indicated that the constitutive expression of ECAT4 sufficiently maintained the undivided state of ES cells even in the absence of LIF.

(3) Removal of ECAT4 expression vector and restoration of differentiation totipotency

The pCAG-IP vector is an expression vector that is maintained extrachromosomally, and is known to disappear when culture is continued in the absence of Pyuguchimycin. Using this, CAG-ECAT4 cells were cultured for 1 month in the absence of pure mouth mycin and in the presence of LIF. The disappearance of foreign gene expression was confirmed by Western plot. These cells differentiated like normal cells after removal of LIF. When CAG-ECAT4 cells were microinjected into blast cells, chimeric mice were formed. Therefore, it was confirmed that even if the cells overexpressing ECAT4 were cultured in the absence of LIF, removal of ECAT4 could regain the totipotency. Since the undifferentiated state of ES cells is sufficiently maintained even in the absence of LIF, the CAG-ECAT4 cells can be used to produce genetically modified animals. Break Variations are easier to make.

Example 7

Identification of DNA recognition sequence using ECAT4

In order to obtain the first clues on the transcriptional regulation by ECAT4, we aimed to determine its DNA recognition sequence by in vitro artificial evolution (SELEX).

(1) SELEX implementation

To prepare a fusion protein consisting of a maltose binding protein and ECAT4, the coding region of the mouse ECAT gene was introduced into pIH1119 (provided from New England and Bio1 abs), and a transgene vector (pIH 11 19—ECAT4) was constructed. This plasmid pIH1119-ECAT4 was introduced into E.coli strain BL21AI (Invitrogen) to obtain a transformant expressing MBP-ECAT4.

Cultivate in 500 ml LB medium under 37 conditions, add I PTG (final concentration 50 M) when the 〇.D. Of the medium reaches 0.3, and incubate for 4 hours at 30 for protein Induction was performed. The cell extract was made up of a protease inhibitor cocktail (Nacalai Tesque) and 5 ml of a lysis buffer (2 OmM Tris-HCl (pH 7.4) containing 0.5 mg / ml of 1 ys0zyme, 20 OmM NaCl, ImM EDTA).

This extract was then subjected to 250 × 1 amylose beads (New Engl and Bio 1 abs) and washed according to the protocol of the product. SELEX was implemented as follows. For the synthesis of double-stranded oligonucleotides, lg SELEX—N2O-01 igo (SEQ ID NO: 11), 1 xg SELEX—N20 RV primer (SEQ ID NO: 12), 2.5 1 10 x Kle no w The reaction was performed in a reaction solution containing buffer, 41 10 mM dNTPs, and 14.7 β1 purified water. Denaturation of the mixture was performed at 95 ° C for 5 minutes, and annealing was performed at 54 ° C for 5 minutes.

A K1 enow fragment (3.611 x 0.225 U in K1enow buffer) was added to the mixture, which was then incubated at 25 ° C for 40 minutes. The double-stranded DNA was purified by phenol Z-cloth form extraction and ethanol precipitation and resuspended in 20 n1 purified water. Performing DNA-protein binding requires 201 D NA, 301 MB P—EC AT 4—binding beads, 201 s 5 × binding buffer (l O OmM HEPES—K〇H, pH 7.9, lmM. EDTA, 1M KC 1, and 50% (Glycerol) and 301 water at 4 ° C. for 30 minutes. Washing of the beads was performed 6 times using 100 1 lysis buffer supplemented with ImM 0 and 0.2 mM PMSF.

DNA was purified by phenol / mouth opening form extraction and ethanol precipitation and resuspended in 201 purified water. PCR amplification was performed using this 51, and the reaction solution at this time was 51 1 OxExTaq buffer, 4/1 1 2.5 mM dNT P, 1 1 30 1 ^ SELEX— It contained N20 FW primer (SEQ ID NO: 13), 1 a1 of 30 MS ELEX-N20 RV primer, 0 of ExTad polymerase and 33.75 L of water. The amplification product was purified using a Micro-bio spin column (BioRad), concentrated to 201 by ethanol precipitation, and half of it was advanced to the next round. This procedure was repeated five times for concentration.

The amplification product in the final eluate of the oligonucleotide that specifically binds to the MBP-ECAT4 fusion protein thus obtained was ligated into a PCR2.1 vector (Invitrogen). Forty clones were randomly selected and sequenced.

(2) Sequence analysis

As described above, we expressed maltose binding protein (MBP) -ECAT4 fusion protein in E.coli and bound them to amylose resin. A 20-mer random oligonucleotide with an adapter sequence at each end was applied to the MBP-ECAT4 binding resin and washed thoroughly. Next, the DNA bound to the resin was amplified by PCR using a primer corresponding to the adapter sequence. The amplification product was applied to a new MBP-ECAT4 column. This procedure was repeated five times to enrich for oligonucleotides that specifically bind to the MBP-ECAT4 fusion protein. The amplification product obtained from the final reaction was subcloned into the pCR2.1 vector for sequence analysis.

And by sequence analysis of 37 randomly picked clones The consensus sequence (G / C) (A / G) (G / C) C (GZC) A TTAN (GZC) was identified (FIG. 6 (A;), SEQ ID NO: 4).

By searching for the flanking sequence of the marker gene used in this study, an ECAT4 consensus sequence was identified approximately 4 kb upstream of the transcription start site in the 5'-flanking region of the mouse GATA6 gene (Fig. 6 (B), sequence number 5). This region is known to be essential for the expression of GAT A6 in the heart (Molkintineta 1., 2000; Sun-Wadaeta 1., 2000). Comparing the genomic sequences of the mouse and human GATA6 genes, this region was found to be highly conserved. The human sequence also contained the ECAT4 consensus (Figure 6 (B), SEQ ID NO: 6).

Example 8

Identification of E CAT 4 Enhancer

In order to elucidate the molecular mechanism by which the mouse ECAT4 gene is specifically expressed in ES cells, an Enhansa analysis using a Lucifera gene receptor gene was performed. A genomic region of about 35 kb containing the mouse ECAT4 gene was divided into eight fragments shown in FIG. 7 (A) and amplified. This was inserted into the repo overnight gene shown in FIG. 7 (B). The repo overnight gene was introduced into ES cells, and luciferase activity was measured 24 hours later. As a result, only fragment D (SEQ ID NO: 7) showed transcriptional activity in ES cells. On the other hand, none of the fragments showed transcriptional activity in the differentiated cells, NIH3T3 cells. Therefore, fragment D was considered to be an ES cell-specific enhancer.

As a result of analysis of the ECAT4 enhancer region, a region having high ES cell-specific activity was found in the region of -6066 / -2292 (fragment D, SEQ ID NO: 7). As a result of examining which part was involved in activation, as shown in Fig. 8 (A), it was active in the region of -4886 / -3444, and among them, the ES cell was in the region of -4737 / -4387. The specific activity was found to be the highest. -In the region of 4737 / _4387, as shown in Fig. 8 (B), -4710 / -4703 contains the binding sequence of the transcription factor LE F / TCF1, which is downstream of the WNT signal. -Deletion of the 4737 / -4611 region reduced its activity, suggesting that this region is important for ECAT4 transcriptional activation. -4524 / -4516 has a STAT3 binding sequence downstream of the LIF signal However, the addition of point mutations reduced the activity, which was again considered to be important for the transcriptional activity of ECAT4. Immediately thereafter, -4515 / -4508 also contained a LEF / TCF1 binding sequence, suggesting that ECAT4 is involved in transcriptional regulation. In addition, since the activity is reduced even when the region of -4497 / -4387 is deleted, this region was considered to be important for the transcriptional activation of ECAT4. Industrial applicability

According to the present invention, an ES cell in which ECAT4 is forcibly expressed is provided. Furthermore, the present invention also provides a consensus sequence for ECAT4, an ECAT4 enhancer, and uses thereof. Since the function of ECAT4 as a transcription factor promoting self-renewal of ES cells has been clearly demonstrated, they are extremely useful in the research and development of ES cells. Sequence listing free text

The base sequence described in SEQ ID NO: 4 is a consensus sequence recognized by ECAT4. The base sequence described in SEQ ID NO: 11 is SELEX-N20-Oiigo. The base sequence described in SEQ ID NO: 12 is a SE LEX-N20RV primer. The base sequence described in SEQ ID NO: 13 is a SELEX-N20 FW primer.

Claims

The scope of the claims

1. A self-renewal determinant of embryonic stem (ES) cells consisting of ECAT4.

2. The ES cell self-renewal determinant according to claim 1, wherein ECAT4 is mouse ECAT4, human ECAT4 or monkey ECAT4.

3. The monkey ECAT4 gene containing the DNA described in (a) or (b) below:

(a) a DNA consisting of the base sequence represented by SEQ ID NO: 10,

(b) a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 10 under stringent conditions and has an ability as a self-renewal determinant of ES cells.

4. Mammalian stem cells in which ECAT4 is forcibly expressed.

5. The mammalian stem cell according to claim 4, wherein the stem cell is an ES cell.

6. The cell according to claim 4, wherein the ECAT4 is mouse ECAT4, human ECAT4, or monkey ECAT4.

7. An antisense polynucleotide comprising a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide represented by SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a part thereof.

8. An agent for promoting cell differentiation, comprising the antisense polynucleotide according to claim 7.

9. ECAT4

10. The ECAT4 enhancer according to claim 9, comprising a part or all of the nucleotide sequence represented by SEQ ID NO: 7.

1 1. SEQ ID NO: 17 The ECAT4 enhancer according to claim 9, comprising the nucleotide sequence represented by SEQ ID NO: 18 or SEQ ID NO: 19.

12. The ECAT4 enhancer according to claim 11, which is a part of the nucleotide sequence represented by SEQ ID NO: 15, and further comprises the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

13. The ECAT4 enhancer according to claim 9, which comprises the nucleotide sequence represented by SEQ ID NO: 15.

14. The ECAT4 enhancer according to claim 13, which is a part of the nucleotide sequence represented by SEQ ID NO: 14 and contains the nucleotide sequence represented by SEQ ID NO: 15.

15. The base sequence of ECAT4 enhancer according to any one of claims 10 to 14, wherein the base sequence has one or more bases deleted, substituted or added, and The ECAT 4 enhancer according to claim 9, which has an ability to promote transcription.

16. Have the ability to hybridize under stringent conditions to DNA comprising the nucleotide sequence of ECAT4 enhancer according to any one of claims 10 to 14, and to promote the transcription of the ECAT4 gene. The ECAT 4 enhancer according to claim 9, characterized in that:

17. An EC AT4 promoter region, comprising the EC AT 4 enhancer according to any one of claims 9 to 16.

18. Consensus sequence recognized by ECAT4.

19. The consensus sequence according to claim 18, comprising a DNA according to any one of the following (a) to (c):

(a) a DNA consisting of the nucleotide sequence of SEQ ID NO: 4,

(c) a DNA capable of hybridizing with a DNA consisting of the nucleotide sequence of SEQ ID NO: 4 under stringent conditions and binding to ECAT4.

20. The consensus sequence according to claim 18, characterized in that it comprises a DNA described in any of the following (a) to (c):

(b) the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16 consisting of a base sequence in which one or more bases have been deleted, substituted or added, and which has an ability to bind to ECAT 4; Having DNA,

(c) D consisting of the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 16 DNA that hybridizes with NA under stringent conditions and has the ability to bind to ECAT4.

21. It comprises a sensor according to any one of claims 9 to 16, a promoter region according to claim 17, or a consensus sequence according to any one of claims 18 to 20. Recombinant vector.

22. A transformed cell transformed with the recombinant vector according to claim 21.

23. A method for screening a substance having an action of controlling the activity of an ECAT4 enhancer or an ECAT4 promoter region, comprising the following steps (a), (b) and (c):

(a) Contacting a test substance with a cell transformed with a DNA in which the ECAT4 enhancer according to any one of claims 9 to 16 or the ECAT4 promoter region according to claim 17 is linked to a reporter gene. The process of

(c) a step of selecting a test substance that controls the activity of the ECAT4 enhancer or ECAT4 promoter region based on the comparison result in (b) above.