JP4268169B2 - Determinants of self-renewal of embryonic stem cells - Google Patents

Description

  The present invention relates to mammalian cells in which ECAT4 is forcibly expressed, ECAT4 enhancer, consensus sequences recognized by ECAT4, and uses thereof.

Embryonic stem cells (ES cells) derived from the inner cell population of the mammalian blastocyst grow rapidly and indefinitely while retaining the ability to differentiate into pluripotent, ie, many types of cells. This property of ES cells is maintained by symmetric self-replication, which results in two identical stem cell daughter cells during cell division. Because of these two kinds of properties, the possibility of a new regenerative medicine that uses tissue to create tissues and cells using ES cells and attracts attention is attracting attention. However, human ES cells also have ethical issues because they must destroy human embryos for ES cell production.
One solution to avoiding such ethical problems is to produce pluripotent cells directly from somatic cells and other cells. The first step in reaching this goal is the identification of a master gene that functions specifically in pluripotent cells and determines cell characteristics.
So far, two types of transcription factors, STAT3 and Oct3 / 4, have been found to be important for mouse ES cells to self-replicate (Burdon et al., (1999). Cells Tissues Organs 165). 131-43., Niwa, (2001). Cell Struct Funct 26, 137-48.), None satisfying the requirements of the master gene has been found.
STAT3 is activated by leukemia inhibitory factor (LIF), which is a kind of cytokine essential for maintaining pluripotency (Smith et al., (1988). Nature 336, 688-90, Williams et al., (1988). Nature 336, 684-7.). Activated STAT3 alone supports self-replication in the absence of LIF (Matsuda et al., (1999). Embo J 18, 4261-9.). Gene targeting experiments have also shown the importance of STAT3 for self-renewal of ES cells (Raz et al., (1999). Proc Natl Acad Sci USA 96, 2846-51.). However, STAT3 expression is found in a wide range of cell types, and some cells reversely induce differentiation (Nakajima et al., (1996). Embo J 15, 3651-8.).
Another transcription factor Oct3 / 4, which is considered a key to ES cell self-renewal, is a transcription factor of the POU family and is expressed in pluripotent cells such as ES cells, ectoderm and primitive embryo cells. Disruption of the mouse Oct3 / 4 gene results in death of the early embryo shortly after implantation, and the inner cell mass of the blastocyst lacking Oct3 / 4 is not pluripotent and only differentiates into the trophoblast lineage. Niwa et al. Confirmed this in ES cells and showed that by suppressing Oct3 / 4, autonomous differentiation into trophoblasts occurred (Niwa et al., (2000). Nat Genet 24, 372-6.). . When these data are combined with the specific expression pattern, it is possible that Oct3 / 4 is a master gene for pluripotent cells.
However, ES cells constitutively expressing Oct3 / 4 from an exogenous promoter still require LIF for self-renewal, and even if adequate amounts of Oct3 / 4 expression are seen, removal of LIF As a result, differentiation into primitive endoderm occurs (Niwa et al., (2000). Nat Genet 24, 372-6.).

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.
By activating the transcription or function of self-replicating determinants that are major for ES cells, it is expected to improve the efficiency and economics of ES cell culture. In addition, by suppressing the transcription or function of the factor, it can be expected that the undifferentiated cells are surely eliminated when the ES cells are induced to differentiate into specific functional cells. The risk of appearance of differentiated cell-derived tumor cells (teratomas) can be avoided.
Therefore, the major self-renewal determinants for ES cells are found, and enhancers that regulate the transcription or function of these factors are used for the search for substances that have the ability to induce or suppress the differentiation of ES cells, and for the research and development of ES cells. Very useful.
The present inventor has already found the ECAT4 gene as a specific expression gene of ES cells. As a result of extensive studies on the ECAT4 gene, it was confirmed that when the mouse ECAT4 gene of ES cells is targeted for destruction, it becomes a natural exogenous endoderm lineage that expresses a high level of endoderm marker. The ECAT4-deficient blastocyst died shortly after implantation, and the inner cell mass of the ECAT4-deficient blastocyst was in vitro and hardly proliferated. In addition, ES cells in which ECAT4 gene was forcibly expressed in ES cells were found to maintain an undifferentiated state even when cultured in the absence of LIF. Therefore, ECAT4 is a major self-renewal determinant for ES cells. I found out.
In addition, the inventor has identified consensus sequences to which ECAT enhancer and ECAT4 bind. In addition, the ECAT4 consensus sequence to which the ECAT4 protein binds is present 4 kb upstream of the transcription initiation site of the 5′-flanking region of the mouse GATA6 gene, and this region is good in the gene sequences of the mouse and human GATA6 genes. Confirmed that it was saved.
The present invention has been completed based on these findings.
That is, the gist of the present invention is as follows.
Item 1. Determinants of self-renewal of embryonic stem (ES) cells consisting of ECAT4,
Item 2. Item 2. The ES cell self-renewal determinant according to Item 1, wherein ECAT4 is mouse ECAT4, human ECAT4 or monkey ECAT4,
Item 3. The monkey ECAT4 gene comprising the DNA described in the following (a) or (b):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 10,
(B) a DNA that hybridizes with a DNA comprising the nucleotide sequence represented by SEQ ID NO: 10 under stringent conditions and has the ability as a self-replicating determinant of ES cells;
Item 4. Mammalian stem cells in which ECAT4 is forcibly expressed,
Item 5. Item 5. The mammalian stem cell according to Item 4, wherein the stem cell is an ES cell.
Item 6. Item 4. The cell according to Item 4 or 5, wherein ECAT4 is mouse ECAT4, human ECAT4 or monkey ECAT4,
Item 7. An antisense polynucleotide comprising a base sequence complementary to or substantially complementary to the base sequence of the polynucleotide represented by SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, or a part thereof;
Item 8. Item 7. A cell differentiation promoter comprising the antisense polynucleotide according to Item 7,
Item 9. ECAT4 enhancer,
Item 10. The ECAT4 enhancer according to Item 9, comprising part or all of the base sequence represented by SEQ ID NO: 7,
Item 11. The ECAT4 enhancer according to Item 9, comprising the base sequence represented by SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19,
Item 12. The ECAT4 enhancer according to Item 11, which is a part of the base sequence represented by SEQ ID NO: 15 and contains the base sequence represented by SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19,
Item 13. The ECAT4 enhancer according to Item 9, which contains the base sequence represented by SEQ ID NO: 15,
Item 14. 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 the ECAT4 enhancer according to any one of Items 10 to 14, comprising a base sequence in which one or more bases have been deleted, substituted or added, and having the ability to promote transcription of the ECAT4 gene Item 10. The ECAT4 enhancer according to Item 9,
Item 16. Item 15. The ECAT4 according to Item 9, which hybridizes with a DNA comprising the nucleotide sequence of the ECAT4 enhancer according to any one of Items 10 to 14 under stringent conditions and promotes transcription of the ECAT4 gene. Enhancer,
Item 17. An ECAT4 promoter region comprising the ECAT4 enhancer according to any one of Items 9 to 16,
Item 18. A consensus sequence recognized by ECAT4,
Item 19. The consensus sequence according to Item 18, which comprises the DNA described in any of the following (a) to (c):
(A) DNA consisting of 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 having the ability to bind to ECAT4;
(C) DNA capable of hybridizing under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 4 and binding to ECAT4;
Item 20. The consensus sequence according to Item 18, which comprises the DNA described in any of the following (a) to (c):
(A) DNA comprising the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16,
(B) DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16, and having the ability to bind to ECAT4 ,
(C) DNA capable of hybridizing under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16 and binding to ECAT4;
Item 21. A recombinant vector comprising 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 screening method for a substance having an action of controlling the activity of the ECAT4 enhancer or ECAT4 promoter region, comprising the following steps (a), (b) and (c):
(A) contacting the test substance with a cell transformed with the DNA in which the ECAT4 enhancer according to any one of Items 9 to 16 or the ECAT4 promoter region according to Item 17 is linked to a reporter gene;
(B) measuring the activity of the reporter gene in the cell contacted with the test substance and comparing the expression level with the activity in the control cell 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 (b) above,
Item 24. 24. ES cell into which the pCAG-IP vector into which the ECAT4 gene has been introduced is introduced Item 25. A genetically modified animal obtained by injecting the ES cell according to Item 24.

FIG. 1 shows the identification of ECAT4.
(A) Comparison of mouse Nkx2.3, mouse ECAT4 and FLJ12581 considered to be a human homologous protein thereof. (B) RT-PCR analysis showing expression profiles of mouse ECAT4, Oct3 / 4 and NAT1. 1. 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 ES cells; Brain; 7. Heart; 8. 8. kidney; Testis; 10. 11. Spleen; Muscle; 12. 13. lungs; Stomach; 14. Ovary; 15. Thymus; 16. Liver; 17. Skin. (C) Western blot analysis showing ECAT4 expression in RF8, J1, CGR8 and MG1.19 ES cell lines. Extracts from ES cells kept undifferentiated (U) or treated with RA for 5 days (D) were analyzed. Western blot was performed using anti-ECAT4 serum or anti-CDK4 antibody.
FIG. 2 shows the targeted disruption of the mouse ECAT4 gene.
(A) Mouse ECAT4 gene, targeting vector, and gene structure after homologous recombination. The positions of probes and restriction enzymes used for Southern blots are shown. Arrows indicate primers for PCR analysis. (B) Southern blot using a 3′-probe produced an 11 kb band from the wild type locus, a 15.4 kb band from the β-geo target locus, and a 7.3 kb band from the hygr target locus. . (C) PCR resulted in a 3.2 kb band from the wild type locus, an 8.5 kb band from the β-geo target locus, and a 4.2 kb band from the hygr target locus. (D) Northern blot confirmed that no ECAT4 transcript was present in the knockout cells.
FIG. 3 shows analysis of ECAT4-deficient ES cells.
(A) Morphology of wild type RF8 cells grown on STO feeder cells (top). ECAT4 cells grown on STO feeder cells (middle) and ECAT4 deficient cells grown without feeder cells (bottom). (B) Growth curve. (C) Northern blot analysis for marker genes. (D) RT-PCR analysis for marker genes.
FIG. 4 shows analysis of preimplantation embryos.
(A) Eight cell stage embryos and blastocysts obtained from wild type females and heterozygous males were stained with X-gal. (B) Blastocysts obtained by heterozygous hybridization were grown on a gelatin-coated petri dish using ES cell medium. The morphology was recorded after 10 days and genotyped by PCR.
FIG. 5 shows forced expression of ECAT4 from a constitutive promoter.
(A) PCR-RT analysis showing expression levels of ECAT4, Oct3 / 4 and NAT1 in MG1.19 cells transformed with parental plasmid or CAG-ECAT4. Cells were grown in the presence or absence of LIF. (B) Constitutive expression of exogenous ECAT4 was confirmed by Western blot. (C) Morphology of control (left) and CAG-ECAT4-expressing cells (right) grown in the presence (top) or absence (bottom) of LIF. (D) Cell Proliferation Rate FIG. 6 shows the transcriptional regulation of ECAT4.
Sequence logo showing the consensus sequence for ECAT4 binding obtained by SELEX (Schneider and Stephens, 1990). Generation of the sequence logo was performed 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 reporter gene containing this region was constructed.
FIG. 7 relates to an enhancer region analysis of the mouse ECAT4 gene.
(A) Mouse ECAT4 gene structure and DNA fragment used for enhancer analysis. (B) Reporter gene structure used for enhancer analysis. Each gene fragment of FIG. 7A was inserted into the 3 ′ end of a reporter gene comprising a thymidine kinase gene promoter, luciferase cDNA, and a poly A addition sequence.
FIG. 8 relates to the enhancer region analysis of the mouse ECAT4 gene.
(A) Fragment region used for enhancer analysis (left) and activity of the fragment (right).
(B) The enhancer region (-4737 / -4387 region) of the mouse ECAT4 gene.

Hereinafter, in this specification, indications with abbreviations such as amino acids, (poly) peptides, (poly) nucleotides, etc. are defined in IUPAC-IUB [IUPAC-IUB Communication on Biological Nomenclature, Eur. J. et al. Biochem. 138: 9 (1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office) and conventional symbols in this field.
The “ES cell self-renewal determinant” of the present invention is a master gene involved in self-replication ability such that embryonic stem cells (ES cells) and the like replicate the same cells without differentiation. This self-replicating determinant of ES cells can be used as a marker for cells having self-replicating ability such as ES cells. By examining the expression of this factor, whether the examined cells have self-replicating ability, Or it can test | inspect whether it is an ES cell. By compulsorily expressing this factor, it is possible to safely maintain the proliferative properties of the cells while maintaining the totipotency of ES cells in the absence of feeder cells and in the absence of cytokines such as LIF. Thereby, the improvement of the efficiency and economical efficiency of ES cell culture can be expected. In addition, since there is no risk of contamination with infectious substances such as endogenous cells derived from feeder cells, which is a concern in clinical application, an improvement in safety can be expected.
In order to induce differentiation of ES cells into specific functional cells, known methods such as homologous recombination, antisense, RNAi (RNA inhibitor), etc. ) To suppress the expression of this self-renewal determinant of ES cells, it can be expected that undifferentiated cells will be eliminated more reliably than before. Thus, further safety improvement can be expected by avoiding the risk of appearance of undifferentiated cell-derived tumor cells (teratomas), which is a concern when clinically applied.
The “ECAT4” of the present invention is natural ECAT4 or genetically modified ECAT4 and may be derived from mammals. For example, mouse ECAT4, rat ECAT4, monkey ECAT4, human ECAT4, etc. can be mentioned.
In addition, the “gene” or “DNA” is equivalent not only to the “gene” or “DNA” represented by the specific base sequence (SEQ ID NOs: 4 to 10, 14 to 19) but also to the function (activity). Or in the case of a gene encoding a protein, the “gene” or “DNA” that encodes its homologues, derivatives and variants is limited to the same biological function. Is included. The “gene” or “DNA” encoding these homologues (homologues), derivatives and mutants is specifically represented by any one of the aforementioned SEQ ID NOs: 4 to 10 under stringent conditions. Examples thereof include “gene” or “DNA” having a base sequence that hybridizes with a specific base sequence.
Stringent conditions are shown in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques in Enzymology, Vol. 152, Ademic Press, San Diego CA) It can be determined based on temperature (Tm). For example, as washing conditions after hybridization, the conditions of about “1 × SSC, 0.1% SDS, 37 ° C.” can be 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, the more stringent hybridization conditions are about “0.5 × SSC, 0.1% SDS, 42 ° C.”, and the more stringent hybridization conditions are “0.1 × SSC, 0.1% SDS, A cleaning condition of about “65 ° C.” can be mentioned. Specifically, as such a complementary strand, a strand consisting of a base sequence that is completely complementary to the base sequence of the target positive strand, and 70% or more, preferably 80% or more, more preferably Examples thereof include a chain composed of a base sequence having a homology of 90% or more, more preferably 95% or more.
In addition, a gene encoding a homologue of the specific base sequence (SEQ ID NOs: 4 to 10, 14 to 19), for example, a gene of another species corresponding to a human gene, is homogene (http: // www) .Ncbi.nlm.nih.gov / HomoloGene /). Specifically, a specific human base sequence is matched with BLAST (Proc. Natl. Acad. Sci. USA., 90: 5873-5877, 1993, http://www.ncbi.nlm.nih.gov/BLAST/). Obtaining the accession number of the sequence (Score is the highest, E-value is 0 and Identity is 100%), and the other accession gene and human gene obtained by entering the accession number into HomoLoGene Can be selected from the list showing the correlation of the gene homologues. In addition, the said gene or DNA does not ask | require the distinction of a functional region, For example, an expression control region, a coding region, an exon, or an intron can be included.
When using the term “ECAT4 gene” or “ECAT4 DNA” in the present specification, unless otherwise specified, the ECAT4 gene (DNA) (SEQ ID NOs: 8 to 10) represented by a specific nucleotide sequence, homologues thereof, It is used to include genes (DNA) encoding mutants and derivatives. Specifically, human ECAT4 gene described in SEQ ID NO: 9 (GenBank Accession No. AB093576), mouse ECAT4 gene described in SEQ ID NO: 8 (GenBank Accession No. AB093574), monkey ECAT4 described in SEQ ID NO: 10. Genes, and rat homologues are included.
In the present specification, “polynucleotide” is used to include both RNA and DNA. The DNA includes any of cDNA, genomic DNA, and synthetic DNA. The RNA includes total RNA, mRNA, and synthetic RNA.
In this 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 biological Fragments, homologues (homologs and splice variants), mutants, derivatives, amino acid modifications and the like are included as long as their functional functions are equivalent. Here, examples of homologs include proteins of other species such as mice and rats corresponding to human proteins, and these were identified by HomoLoGene (http://www.ncbi.nlm.nih.gov/HomoloGene/). It can be identified a priori from the base sequence of the gene. Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having amino acid sequences modified by artificial deletions, substitutions, additions and insertions. . Examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and still more preferably 97% homologous to a protein or (poly) peptide without mutation. The amino acid modifications include naturally occurring amino acid modifications and non-naturally occurring amino acid modifications, and specific examples include phosphorylated amino acids.
Accordingly, when the term “ECAT4 protein” or simply “ECAT4” is used in the present specification, ECAT4 represented by a specific amino acid sequence (SEQ ID NOs: 1 to 3) and homologues, variants and derivatives thereof unless otherwise specified. And amino acid modifications. Specifically, human ECAT4 having the amino acid sequence set forth in SEQ ID NO: 2, mouse ECAT4 having the amino acid sequence set forth in SEQ ID NO: 1, monkey ECAT4 having the amino acid sequence set forth in SEQ ID NO: 3, and their Rat homologues and the like are included.
Hereinafter, specific contents of the present invention will be described.
(1) Mammalian stem cells in which ECAT4 is forcibly expressed
The “mammalian stem cell in which ECAT4 is forcibly expressed” of the present invention refers to 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 and rats, and may be primary cells taken from individuals or cultured cells. What is necessary is just a cell derived from. Mammalian cells are, for example, commercially available cultured cells (such as ATCC), mouse ES cells, monkey ES cells and human ES cells, and more preferably mouse ES cells, monkey ES cells that differentiate in the absence of LIF, Embryonic stem cells such as human ES cells are not limited to these.
In order to forcibly express ECAT4, for example, an expression vector containing the ECAT4 gene may be introduced into the cell. Specific examples of the method of introducing the expression vector include the calcium phosphate method, the DEAE-dextran method, the electroporation method, the gene Known methods such as a method using a lipid for introduction (Lipofectamine, Lipofectin; Gibco-BRL), a microinjection method and the like can be mentioned.
An expression vector containing the ECAT4 gene can be prepared based on ordinary genetic engineering techniques. The expression vector used here can be appropriately selected according to the host to be used, the purpose, etc., and includes plasmids, phage vectors, virus vectors and the like.
Examples thereof include plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1, pRc / RSV, and pRc / CMV, and viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors.
The vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a selection marker gene, and a terminator. Moreover, it may have a gene encoding a fusion protein of marker protein and ECAT4.
Here, the “marker gene” is a gene with a clear phenotype useful for genetic analysis, and its expression can be directly observed after gene introduction. For example, genes for fluorescent proteins such as GFP and GFP mutants such as CFP and YFP can be mentioned, but not limited to this, genes can be expressed in living cells by introducing genes into cells and expressing them. Anything that can be confirmed to be introduced is included in the category of the marker gene of the present invention. The marker protein is a protein encoded by a marker gene.
By introducing a vector to which such a marker gene is added, cells expressing ECAT4 can be easily detected. Furthermore, since only stem cells that express ECAT4 can be selected by using ES cells into which such a gene has been introduced, undifferentiated cells and differentiated cells can be selected when ES cells are differentiated into functional cells. It can also be used as a marker. This avoids the risk of transplanting undifferentiated cells, which are at risk of teratoma formation, into the living body.
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 target gene is expressed in the presence of tetracycline and not expressed in the non-coexistence can be given (Niwa et al., (2000). Nat Genet 24, 372-6.). Since cells into which these vectors have been introduced can switch on / off ECAT4-expressing cells depending on the culture conditions, ECAT4 is stably forcibly expressed between ES cells, and expression is shut off when differentiation is induced. It is considered that differentiation can be efficiently induced 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. Specific examples include a pCAG-IP vector. ES cells into which an ECAT4 expression vector having such properties has been introduced are very useful for preparing genetically modified animals such as transgenic mice. Since the ES cell into which the vector has been introduced disappears from the cell under certain conditions, it is easier and more reliable than controlling the expression level of ECAT4 using a method in which the vector remains in the cell, such as regulated promoter. Modified animals can be made.
For example, since CAG-ECAT4 cells prepared using a pCAG-IP vector containing ECAT4 gene can be cultured in the absence of LIF or feeder cells, the desired target gene is expressed in CAG-ECAT4 cells. The operation of introducing other vectors for knocking out, knocking down or knocking in is very easy.
Here, the genetically modified animal is an animal in which the standardized 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 base sequence complementary to or substantially complementary to the base sequence of the ECAT4 gene or a part thereof, and has an action capable of suppressing the expression of the ECAT4 gene As long as it is, antisense RNA and antisense DNA are included.
Here, the substantially complementary base sequence is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more, most preferably, the base sequence complementary to the base sequence of the ECAT4 gene. Includes a base sequence having a homology of about 95% or more. In particular, it is about 70% or more, preferably about 80% or more, more preferably about 90%, with the complementary strand of the base sequence encoding the N-terminal part of the base sequence of the ECAT4 gene (eg, the base sequence near the start codon). Antisense polynucleotides with a homology of at least%, most preferably at least about 95% are preferred. Specific examples include antisense polynucleotides having a sequence complementary to or substantially complementary to the base sequence described in any of SEQ ID NOs: 8 to 10, or a part thereof.
A polynucleotide having a complementary base sequence (complementary strand, reverse strand) is a full-length sequence of a polynucleotide comprising the base sequence shown in each of the above SEQ ID NOs, or a base sequence having a base length of 15 bases at least in the base sequence Means a polynucleotide having a base-complementary relationship based on a base pair relationship such as A: T and G: C with respect to a partial sequence having the above (for convenience, these are also referred to as “positive strands”) To do. However, such a complementary strand is not limited to the case where it forms a completely complementary sequence with the target positive strand base sequence, but has a complementary relationship that allows it to hybridize with the target positive strand under stringent conditions. You may have.
Further, the polynucleotide on the positive strand side includes not only those having the base sequence of the ECAT4 gene or a partial sequence thereof, but also a strand comprising a base sequence that is more complementary to the base sequence of the complementary strand. 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. In order to prevent degradation by a hydrolase such as nuclease, the phosphate residues (phosphates) of each nucleotide constituting the antisense DNA are changed to chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. May be substituted. These antisense polynucleotides can be produced using a known DNA synthesizer.
Such an antisense polynucleotide can hybridize with the RNA of the ECAT4 gene, inhibit the synthesis or function of the RNA, or regulate the expression of the ECAT4 gene through the interaction with the RNA. Can be controlled.
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. 5 'end hairpin loop of protein gene, 5' end 6-base pair repeat, 5 'end untranslated region, polypeptide translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 ' An end palindromic region or a 3 ′ end hairpin loop or the like can be selected as a preferred region of interest, but any region within a protein gene can be selected as a 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 to the target region, the target nucleic acid It can be said that it is “antisense”.
Antisense polynucleotides include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, non-polynucleotides, Other polymers with a nucleotide backbone (eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or other polymers containing special linkages (provided that the polymer is a base such as found in DNA or RNA) And a nucleotide having a configuration allowing the attachment of a pairing or base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, Null peptide, poly-L-lysine, etc.) and sugars (eg, monosaccharides), etc., side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, , Metals, radioactive metals, boron, oxidizing metals, etc.), alkylating agents, and modified bonds (eg, alpha anomeric nucleic acids) Also good. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only 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, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted. The antisense polynucleotide of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acids include nucleic acid sulfur derivatives, thiophosphate derivatives, polynucleoside amides and oligonucleoside amides that are resistant to degradation.
The antisense polynucleotide of the present invention can be designed, for example, as follows. That is, to make the antisense polynucleotide in the cell more stable, to increase the cell permeability of the antisense polynucleotide, to increase the affinity for the target sense strand, and to reduce the toxicity In some cases, the antisense polynucleotide is less toxic. Many such modifications have been reported in, for example, Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Applications, CRC Press, 1993, and the like.
The antisense polynucleotide of the present invention may be provided in a special form such as a liposome or a microsphere, or may be provided in an added form adapted by gene therapy. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). Such can be attached to the 3 'or 5' end of the nucleic acid and can be attached via a base, sugar, intramolecular nucleoside bond. Examples of the other group include a cap group specifically arranged at the 3 ′ end or 5 ′ end of a nucleic acid, which prevents degradation by a nuclease such as exonuclease or RNase. 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.
The antisense polynucleotide of the present invention may be double-stranded as described above, and binds to RNA encoding ECAT4 and destroys the RNA or suppresses its function. That is, a part of RNA encoding ECAT4 and double-stranded RNA containing RNA complementary thereto are also included in the antisense polynucleotide of the present invention.
The use of the antisense polynucleotide will be described in detail. For example, by administering an antisense oligonucleotide or a chemical modification thereof directly to a target cultured cell or tissue in the same manner as in this type of gene therapy. It can be carried out by a method for controlling the expression of the target gene. The antisense polynucleotide of the present invention can also be administered to cells as it is or by incorporating it into a gene therapy vector. Also in these cases, the dose and administration method vary depending on the cell type, the number of cells, etc., and can be appropriately selected by those skilled in the art.
An antisense polynucleotide or a chemical modification thereof can bind to a sense strand mRNA in a cell to control the expression of a target gene, that is, the expression of ECAT4, and thus control the function (activity) of ECAT4. Can do.
In the method of directly administering an antisense polynucleotide or chemical modification thereof to cultured cells, the antisense polynucleotide or chemical modification thereof used is preferably 10 to 1000 bases, more preferably 15 to 500 bases, most preferably May have a length of 16 to 30 bases. In the administration, antisense oligonucleotides or chemical modifications thereof can be formulated (reagentized) using commonly used stabilizers, buffers, solvents and the like.
Examples of the method using the above recombinant virus include, for example, the anti-viral polypeptide of the present invention in a virus genome such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus, vaccinia virus, poliovirus, and synbisvirus. An example is a method of incorporating nucleotides into a living body. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses and the like are particularly preferred. Examples of non-viral introduction methods include the liposome method and the lipofectin method, and the liposome method is particularly preferable. Other non-viral introduction methods include, for example, a microinjection method, a calcium phosphate method, an electroporation method, and the like.
The cell differentiation promoting agent of the present invention comprises the above-described antisense polynucleotide or a chemically modified product thereof, a recombinant virus containing these, an infected cell into which these viruses have been introduced, and the like as active ingredients. The cell differentiation promoter of the present invention can be used as a reagent or a medicine. The administration form of the composition can be appropriately determined depending on the target cell or tissue. The medicament or reagent of the present invention can be, for example, in a form (such as Sendai virus (HVJ) -liposome) in which a viral vector containing the antisense polynucleotide of the present invention is embedded in liposomes or membrane fusion liposomes. These liposome preparation forms include suspensions, freezing agents, centrifugal concentrated freezing agents, and the like. Moreover, it can also be set as the form of the cell culture solution infected with the virus in which the vector containing the said antisense polynucleotide of this invention was introduce | transduced. The dose of the active ingredient in these various forms of preparation can be adjusted as appropriate according to the purpose. Usually, in the case of an antisense polynucleotide against the ECAT4 gene, about 0.001 μg-100 mg, preferably about 0.001 μg-10 mg may be added / administered once every several days to several months. For example, in the case of a 10 cm petri dish, the antisense nucleotide of the present invention may be added in an amount of 0.0001 μg to 100 mg, preferably 0.001 μg to 10 mg once every several days to several months.
Furthermore, the present invention relates to (i) a double-stranded RNA containing a part of RNA encoding ECAT4 and RNA complementary thereto, (ii) a pharmaceutical comprising the double-stranded RNA, (iii) ECAT4 (Iv) a drug containing the ribozyme, (v) an expression vector containing a gene (DNA) encoding the ribozyme, and the like. Similar to the above antisense polynucleotide, double-stranded RNA, ribozyme, and the like can also destroy RNA transcribed from the DNA of the present invention or suppress its function. It can be used as a reagent or therapeutic agent such as double-stranded RNA or ribozyme capable of suppressing the function of ECAT4 or DNA encoding the same.
Double-stranded RNA can be produced by designing based on a polynucleotide sequence encoding ECAT4 according to a known method (eg, Nature, 411, 494, 2001). The ribozyme can be designed and produced based on the sequence of the polynucleotide encoding ECAT4 according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, 221, 2001). For example, it can be produced by substituting a part of the known ribozyme sequence with a part of RNA encoding ECAT4. Examples of the RNA encoding ECAT4 include a sequence in the vicinity of a consensus sequence NUX (wherein N represents all bases and X represents a base other than G) that can be cleaved by a known ribozyme. When the above double-stranded RNA or ribozyme is used as the above reagent or pharmaceutical, it can be formulated and administered in the same manner as the antisense polynucleotide. The expression vector is used in the same manner as a known gene therapy method, and used as the 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 is a nucleotide sequence that promotes the transcription of ECAT4. If the ECAT4 promoter region containing the ECAT4 enhancer of the present invention is used, transcription of the ECAT4 gene and the like can be performed efficiently.
The ECAT4 enhancer of the present invention is represented by (i) an ECAT4 enhancer characterized by including part or all of the base sequence represented by SEQ ID NO: 7, (ii) represented by SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 ECAT4 enhancer containing a base sequence, (iii) ECAT4 enhancer (iv) which is a part of the base sequence shown by SEQ ID NO: 15 and contains the base sequence shown by SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 (E) an ECAT4 enhancer containing the base sequence represented by SEQ ID NO: 15, (v) an ECAT4 enhancer which is a part of the base sequence represented by SEQ ID NO: 14 and comprises the base sequence represented by SEQ ID NO: 15, (vi) one in the base sequence of the ECAT4 enhancer according to any one of i) to (v) or An ECAT4 enhancer comprising a nucleotide sequence in which several bases are deleted, substituted or added, and having the ability to promote transcription of the ECAT4 gene; (vii) any one of (i) to (v) An ECAT4 enhancer characterized in that it hybridizes with DNA comprising the nucleotide sequence of the ECAT4 enhancer under stringent conditions and has the ability to promote transcription of the ECAT4 gene.
As a specific example of (i), for example, the DNA in the region from the −4737 position to the −4611 position of the base sequence shown in FIG. 8B (the region from the first position to the 127th position in SEQ ID NO: 15) And an ECAT4 enhancer containing DNA in the region of positions −4497 to −4387 (region of positions 241 to 351 in SEQ ID NO: 15) of the base sequence shown in FIG. 8 (B) Can be mentioned.
In the present invention, a “promoter region” is a type of regulatory gene, which is a region on DNA that determines the initiation site of gene transcription and directly regulates its frequency. Contains promoter elements that are subject to cell type-specific or tissue-specific control, or sufficient promoter elements to cause promoter-dependent gene expression induced by an exogenous signal or factor (eg, a transcriptional activation protein) May be.
The “ECAT4 promoter region” of the present invention includes the above-described ECAT4 enhancer of the present invention, and may be any DNA as long as it has ECAT4 promoter activity. Specifically, for example, DNA consisting of a part or all of the base sequence shown in SEQ ID NO: 14 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 the gene and exhibits a transcription control action.
As a consensus sequence recognized by ECAT4 of the present invention,
(I) A consensus sequence recognized by ECAT4, which comprises the DNA described in any of (a) to (c) below:
(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 having the ability to bind to ECAT4;
(C) DNA capable of hybridizing under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 4 and binding to ECAT4;
(Ii) A consensus sequence recognized by ECAT4, which comprises the DNA described in any of (a) to (c) below:
(A) DNA comprising the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16,
(B) DNA comprising a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16, and having the ability to bind to ECAT4 ,
(C) DNA capable of hybridizing under stringent conditions with DNA consisting of the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16 and binding to ECAT4;
(Iii) a consensus sequence recognized by ECAT4 characterized in that it contains a part of the sequence described in (i) or (ii) above,
Can be mentioned. Here, “one or more bases” is preferably 1 to 3 bases, more preferably 1 to 2 bases. The “part of sequence” in (iii) is, for example, DNA of several bases to 10 bases from the 5 ′ end of the base sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16, or 3 ′ side Although DNA of several bases-10 bases from the terminal can be mentioned, it is not limited to this.
The ECAT4 enhancer of the present invention, the consensus sequence recognized by ECAT4, and the ECAT4 promoter region are human or other mammalian cells (eg, human lymphocyte cells, mouse fibroblasts, mouse ES cells, rat ES cells, human ES cells). Alternatively, any of genomic DNA, cDNA, and synthetic DNA derived from any tissue in which those cells exist may be used.
Examples of methods 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 such as a DNA synthesizer model 380A (manufactured by ABI) or the like can be used.
Next, a method for preparing the ECAT4 enhancer or ECAT4 promoter region of the present invention using PCR will be described. A genomic library used as a template is prepared from a tissue of a mammal such as a human or mouse according to a method described in, for example, “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press, etc. can do. Further, commercially available genomic DNA such as human genomic DNA (manufactured by Clontech) or commercially available genomic library such as human genome walker kit (manufactured by Clontech) can be used. Next, PCR is performed 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 side. Also good. The DNA amplified as described above can be obtained from "Molecular Cloning: A Laboratory Manual 2nd edition" (1989), Cold Spring Harbor Laboratory Press, "Current Protocols In Molecular Bios. 1987". It can be cloned into a vector according to the usual method described in ISBN0-471-50338-X or the like. 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 pBluescript II manufactured by Stratagene. The base sequence of the cloned DNA is F.I. Sanger, S.M. Nicklen, A.M. R. Coulson, Proceedings of National Academy of Science U. S. A. (1977), 74, 5463-5467, and the like.
Next, a method for preparing using a hybridization method will be described.
First, the DNA used for the probe is labeled. As DNA used for the probe, DNA having at least a part of the base sequence such as enhancer to be prepared, promoter region, etc., for example, the base sequence represented by any of SEQ ID NOs: 4 to 7 and SEQ ID NOs: 14 to 19 or DNA consisting of a part of the base sequence and having a chain length of 16 to 160 bases, and one or more bases deleted, substituted or added in the base sequence of the DNA Examples include DNA consisting of a base sequence, DNA that hybridizes with the DNA under stringent conditions, and the like.
The DNA used for the probe can be obtained by, for example, the usual DNA preparation methods described above, such as chemical synthesis methods, PCR, and hybridization methods. The DNA used for the probe may itself have the ability to control the transcription of the gene encoding ECAT4.
In order to label the DNA used for the probe with a radioisotope, for example, Random Labeling Kit manufactured by Boehringer or Takara Shuzo can be used, and dCTP in a normal PCR reaction composition is [α- ³² Instead of P] dCTP, labeling can also be performed by performing a PCR reaction using the DNA used for the probe as a template. In addition, when the DNA used for the probe is labeled with a fluorescent dye, for example, ECL Direct Nucleic Acid Labeling and Detection System (manufactured by Amersham Pharmacia Biotech) or the like can be used. As a DNA library for hybridizing the probe, for example, a genomic DNA library derived from an animal such as a rodent such as a mouse can be used. As the DNA library, a commercially available genomic DNA library can be used, and “Molecular Cloning: A Laboratory Manual 2nd edition” (1989), Cold Spring Harbor Laboratory Press and “Current Protocols In Molecular 7 Biomolecules”. ), John Wiley & Sons, Inc. In accordance with a normal library preparation method described in ISBN0-471-50338-X or the like, for example, using λ vectors such as λ FIX II, λ EMBL3, λ EMBL4, λ DASH II manufactured by Stratagene, Gigapack packaging Extractions (Stratagene Etc.) can be used for in vitro packaging and a genomic DNA library can be prepared and used.
Examples of the hybridization method include colony hybridization and plaque hybridization, and the method may be selected according to the type of vector used for preparing the library. For example, when the library used is a library constructed with a plasmid vector, colony hybridization can be performed. Specifically, first, DNA of the library is introduced into a host microorganism to obtain transformed cells, the obtained transformed cells are diluted and spread on an agar medium, and cultured at 37 ° C. until colonies appear. Moreover, when the library used is a library constructed with a phage vector, plaque hybridization can be performed. Specifically, first, host microorganisms and library phages are mixed under conditions allowing infection, and further mixed with a soft agar medium, which is then spread on the agar medium. Thereafter, the cells are cultured at 37 ° C. until plaques appear. More specifically, for example, a method described in Molecular Cloning 2nd edition (written by J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press 1989) 2.60 to 2.65, etc. In accordance with NZY agar medium, agar medium 1mm ² About 9.0 x 10 at a density of 0.1 to 1.0 pfu per ⁵ Spread the pfu phage library and incubate at 37 ° C. for 6-10 hours.
Next, in any of the above-described hybridization methods, a membrane filter is placed on the surface of the agar medium on which the above-described culture is performed, and the transformed cells or phages carrying the plasmid are transferred to the membrane filter. The membrane filter is treated with an alkali, then neutralized, and then subjected to a treatment for fixing DNA to the filter. More specifically, for example, in the case of plaque hybridization, the agar medium according to the usual method described in Cloning and Sequence: Plant Biotechnology Experiment Manual (Watanabe, Sugiura Editing, Rural Bunkasha 1989) and the like. Nitrocellulose filter or nylon filter, for example, Hybond-N ⁺ (Amersham Pharmacia Biotech) is placed and allowed to stand for about 1 minute to adsorb phage particles to the membrane filter. Next, the filter is 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 on the filter. .5M sodium chloride, 0.5M Tris-HCl, pH 7.5) for about 5 minutes. The filter is washed with a washing solution (300 mM sodium chloride, 30 mM sodium citrate, 200 mM Tris-HCl) for about 5 minutes, and then, for example, baked at about 80 ° C. for about 90 minutes to fix the phage DNA to the filter. Hybridization is performed using the thus prepared filter and the probe. Reagents and temperature conditions for performing hybridization are, for example, D.I. M.M. Glover edited by "DNA cloning, a practical approach" IRL PRES (1985) ISBN 0-947946-18-7, Cloning and Sequence: Plant Biotechnology Experiment Manual (Watanabe, Sugiura Editing, Rural Bunkasha 1989), or Molecular Cloning 2nd edition (by J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) and the like. For example, denatured non-specific DNA containing 450-900 mM sodium chloride, 45-90 mM sodium citrate, sodium dodecyl sulfate (SDS) at a concentration of 0.1-1.0% (W / V) In a concentration of 0 to 200 μg / mL, and in some cases albumin, ficoll, polyvinylpyrrolidone and the like may be included in a concentration of 0 to 0.2% (W / V) respectively, preferably A prehybridization solution containing 900 mM sodium chloride, 90 mM sodium citrate, 1% (W / V) SDS and 100 μg / mL denatured Calf-thymus DNA was prepared as described above. ² The filter is prepared at a rate of 50 to 200 μL per unit, and the filter is immersed in the solution and incubated at 42 to 68 ° C. for 1 to 4 hours, preferably at 45 ° C. for 2 hours. Next, for example, 450 to 900 mM sodium chloride, 45 to 90 mM sodium citrate, SDS at a concentration of 0.1 to 1.0% (W / V), A hybridization solution containing 200 μg / mL and optionally containing albumin, Ficoll, polyvinylpyrrolidone, etc. at a concentration of 0 to 0.2% (W / V), preferably 900 mM sodium chloride A hybridization solution containing 90 mM sodium citrate, 1% (W / V) SDS and 100 μg / mL denatured Calf-thymus DNA, and the probe prepared by the above-described method (filter 1 cm ² 1.0 × 10 per ⁴ ~ 2.0 × 10 ⁶ 1 cm of filter solution mixed with cpm equivalent) ² Prepare a 50-200 μL per solution, soak the filter in the solution, and incubate at 42-68 ° C. for 4-20 hours, preferably at 45 ° C. for 16 hours to carry out the hybridization reaction. After the hybridization reaction, the filter is taken out and contains 42 to 68 ° C. containing 15 to 300 mM sodium chloride, 1.5 to 30 mM sodium citrate, 0.1 to 1.0% (W / V) SDS, and the like. 1 to 4 times of filter washing for 10 to 60 minutes with a washing solution at 55 ° C. containing 300 mM sodium chloride, 30 mM sodium citrate, and 1% (W / V) SDS. Preferably, 15 minutes of washing is performed twice. Further, the filter is lightly rinsed with 2 × SSC solution (containing 300 mM sodium chloride and 30 mM 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 on the filter of the DNA hybridized with the probe used. A clone having the DNA can be isolated by identifying a clone corresponding to the position of the detected DNA on the filter on the original agar medium and catching it. Specifically, for example, the filter is exposed to an imaging plate (Fuji Film) for 4 hours, and then the plate is analyzed using BAS2000 (Fuji Film) to detect a signal. The portion corresponding to the position where the signal was detected in the agar medium used for the production of the filter was cut out to about 5 mm square, and this was cut into about 500 μL of SM buffer (50 mM Tris-HCl pH 7.5, 0.1 M sodium chloride, 7 mM magnesium sulfate and 0.01% (W / V) gelatin) for 2-16 hours, preferably 3 hours to elute phage particles. The obtained phage particle eluate was prepared according to the method described in Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) 2.60 to 2.65. Spread on agar medium and incubate at 37 ° C. for 6-10 hours. Using this agar medium, phage DNA is immobilized on a filter in the same manner as described above, and hybridization is performed using this filter and the probe described above. The phage particles are eluted from the portion corresponding to the position where the signal is detected in the agar medium used for the preparation of the filter, spread on the agar medium, and the filter is prepared in the same manner as described above. Do. By repeating such identification and purification of the phage clone, a phage clone containing DNA having a base sequence that hybridizes with the probe used is obtained. The DNA possessed by the clone obtained by screening by hybridization as described above is subcloned into a plasmid vector that is easy to prepare and analyze DNA, such as commercially available pUC18, pUC19, pBLUESCRIPT KS +, pBLUESCRIPT KS-, etc. Preparing plasmid DNA; Sanger, S.M. Nicklen, A.M. R. Coulson, Proceedings of National Academy of Science U. S. A. (1977), 74, 5463-5467, etc., and the base sequence can be determined using the dideoxy terminating method. Preparation of a sample used for base sequence analysis is described in, for example, Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) 13.15. It can be performed according to the primer extension method. In addition, phage clones were prepared according to the method described in Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Harbor Laboratory Press, 1989) 2.60 to 2.65, etc. Amplification in a liquid medium and preparation of a phage solution, from which phage clone DNA is extracted using, for example, Lambda-TRAPPLUS DNA Isolation Kit (manufactured by Clontech), etc. Thus, it is possible to prepare a sample for base sequence analysis and analyze 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, “ability to control transcription” means, for example, an activity for initiating and / or promoting transcription of a gene located downstream of the 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 are prepared by introducing mutations into a sequence containing the base sequence represented by any of SEQ ID NOs: 4-7 and SEQ ID NOs: 14-19. Also good. Specifically, for example, A.I. Greener, M.M. Can be obtained by randomly introducing mutations using the method described in Callahan, Strategies (1994) 7, 32-34, and the like. Kramer, et al. Nucleic Acids Research (1984) 12, 9441; Kramer, H .; J. et al. Gapped duplex method described in Frits, Methods in Enzymology (1987) 154, 350, or the like. A. Kunkel, Proc. of Natl. Acad. Sci. U. S. A. (1985) 82, 488 or T.W. A. Kunkel, et al. , Methods in Enzymology (1987) 154, 367 and the like, can be obtained by introducing a site-specific mutation using the Kunkel method. Alternatively, it can be obtained by preparing a chimeric DNA in which one or several partial base sequences in the ECAT4 promoter region containing the base sequence represented by SEQ ID NO: 15 are replaced with a part of the DNA of another promoter. For example, S.M. Henikoff, et al. Gene (1984) 28, 351, C.I. Yanish-Perron, et al. The method described in Gene (1985) 33, 103 etc. can be used.
The promoter region of the present invention prepared by the various methods described above can be prepared by a conventional method, for example, “Tamura Takaaki (published by Yodosha), New Transcriptional Control Mechanism (2000)” pages 33-40, “Nomura Shintaro In accordance with the method described in Toshiki Watanabe, written by Toshiki Watanabe (published by Shujunsha, Deisotope Experiment Protocol (1998)), etc., it is obtained by deleting a part of the base while maintaining the promoter activity ( That is, DNA prepared by excision using an appropriate restriction enzyme can also be used as the promoter region of the present invention. The ability of the obtained DNA to control the transcription of the ECAT4 gene can be confirmed by the method described later.
In the preparation of the recombinant vector of the present invention, the recombinant vector for inserting the ECAT4 enhancer or ECAT4 promoter region of the present invention (hereinafter referred to as “ECAT4 enhancer / promoter region” in the present specification) functions as a desired cell. Any possible recombinant vector may be used. When using the ECAT enhancer of the present invention, a known appropriate promoter can be linked in a functional form. The recombinant vector of the present invention may contain a marker gene (for example, a kanamycin resistance gene, a hygromycin resistance gene, a neomycin resistance gene, etc.) for selecting cells into which the recombinant vector has been introduced. In the recombinant vector, the gene is located at a position where the enhancer / promoter region of the present invention and the desired gene are operably linked on the recombinant vector, for example, downstream of the site where the enhancer / promoter region is inserted. If there is an insertion site, it can be preferably used for the construction of a recombinant vector for expressing a desired gene in a cell. Here, the gene insertion site is, for example, a base sequence that can be specifically recognized and cleaved by a restriction enzyme usually used in genetic engineering techniques, and is a kind that exists only on a recombinant vector having the enhancer / promoter region of the present invention. These restriction enzyme recognition sequences are preferred. Specific examples of the recombinant vector include pUC plasmids [pUC118, pUC119 (Takara Shuzo) and the like], pSC101 plasmids, pBR322 plasmid (Boehringer Mannheim) and the like.
In the preparation of the recombinant vector of the present invention, the recombinant vector for inserting the consensus sequence recognized by ECAT4 of the present invention may be any recombinant vector that can function in a desired cell. A marker gene (for example, a kanamycin resistance gene, a hygromycin resistance gene, a neomycin resistance gene, etc.) for selecting cells into which the vector has been introduced may be included. In the recombinant vector, a suitable promoter and downstream of the position where the consensus sequence of the present invention and the desired gene are operably linked on the recombinant vector, for example, the site where the consensus sequence is inserted. If there is a gene insertion site, it can be preferably used for the construction of a recombinant vector for expressing a desired gene in cells. Here, the gene insertion site is, for example, a base sequence that can be specifically recognized and cleaved by a restriction enzyme usually used in genetic engineering techniques, and is the only one on a recombinant vector having a consensus sequence recognized by ECAT4 of the present invention. The kind of restriction enzyme recognition sequence present is preferred. Specific examples of the recombinant vector include pUC plasmids [pUC118, pUC119 (Takara Shuzo) and the like], pSC101 plasmids, pBR322 plasmid (Boehringer Mannheim) and the like.
Furthermore, in order to examine the activity of the ECAT4 enhancer / promoter region, or the binding of the consensus sequence recognized by ECAT4 and ECAT4, a detectable structural gene can be linked downstream of the ECAT4 enhancer / promoter region or the consensus sequence recognized by ECAT4. Good. Various reporter genes are used as the structural gene linked downstream of the enhancer / promoter region or consensus sequence. Reporter genes include luciferase gene, CAT (chloramphenicol acetyltransferase) gene, alkaline phosphatase gene, β-galactosidase gene, and any other structural gene. In order to incorporate the structural gene into a vector, an appropriate restriction enzyme cleavage site located downstream of the ECAT4 enhancer / promoter region or the consensus sequence recognized by ECAT4 is used. For example, commercially available vectors such as pGL3 Basic and pGL3 promoter (Promega) may be used.
Examples of the host to be transformed with the above recombinant vector include Escherichia bacteria, Bacillus bacteria, yeast, insect cells, animal cells, and the like. Examples of animal cells include monkey cells COS-7, Vero, Chinese hamster cells CHO (hereinafter abbreviated as CHO cells), dhfr gene-deficient Chinese hamster cells CHO (hereinafter abbreviated as CHO (dhfr-) cells), mouse L Cell, mouse AtT-20, mouse myeloma cell, rat GH3, mouse fibroblast 3T3-L1, human liver cancer cell HepG2 (hereinafter abbreviated as HepG2 cell), human osteosarcoma cell MG-63 (hereinafter MG-63 cell) Abbreviated), human FL cells, white adipocytes, egg cells, ES cells (Evans, MJ and Kaufman, KH (1981) Nature, 292, 154) and the like. Preferred are tissue-derived stem cells or ES cells derived from mammals, and particularly preferred examples include mouse ES cells, rat ES cells, and human ES cells.
Methods for transformation into these cells include calcium phosphate method (Graham et al. (1973) Virology, 52, 456), electroporation method (Ishizaki et al. (1986) Cell Engineering, 5, 577), microinjection method, gene For example, a method using a lipid for introduction (Lipofectamine, Lipofectin; Gibco-BRL) is used. More specifically, in order to transform animal cells, for example, cell engineering separate volume 8 new cell engineering experiment protocol. 263-267 (1995) (published by Shujunsha), Virology, Vol. 52, 456 (1973). The above 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 DNA containing ECAT4 enhancer or ECAT4 promoter region, a compound that controls (activates / suppresses) the activity of ECAT4 enhancer or ECAT4 promoter region by using the above transformant Can be screened. In addition, if the recombinant vector of the present invention is a DNA containing a consensus sequence recognized by ECAT4, a compound that controls (activates / suppresses) the binding between ECAT4 and the consensus sequence by using the transformant described above. It can be used for screening.
(4) A method for screening a substance that controls (activates / represses) the activity of the ECAT4 enhancer or ECAT4 promoter region, and a screening method using a consensus sequence recognized by ECAT4
By using the ECAT4 gene, ECAT4 enhancer, ECAT4 promoter region, or consensus sequence recognized by ECAT4 of the present invention, a substance that controls (activates / suppresses) the expression of ECAT4, or the function of ECAT4 (activates / activates / activates). The substance to be suppressed) can be screened.
For example, a substance that activates or suppresses the expression of ECAT4 can be screened by introducing a chimeric gene in which a luciferase gene is linked to the base sequence of the ECAT4 enhancer / promoter region into a cell (eg, ES cell). Similarly, a substance that affects transcriptional activation or transcriptional repression by ECAT4 by introducing a chimeric gene of a promoter gene containing a consensus sequence recognized by ECAT4 and a reporter gene into a cell (for example, ES cell), Alternatively, a substance having the same activity as ECAT4 can be screened. Furthermore, a substance that inhibits the binding of ECAT4 and the consensus sequence of the present invention can be searched for by a known binding assay, for example, a binding assay using the SPA method (Amersham Pharmacia).
Examples of the screening method for a substance that controls the activity of the ECAT4 enhancer or ECAT4 promoter region of the present invention include 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 reporter gene;
(B) measuring the activity of the reporter gene in the cell contacted with the test substance and comparing the expression level with the activity in the control cell not contacted with the test substance;
(C) A step of selecting a test substance that controls the activity of the ECAT4 enhancer or the ECAT4 promoter region based on the comparison result of (b) above.
As the cells used in such screening, the transformant of the present invention described in the above (3) is useful.
Test substances (candidate substances) to be screened by the screening method of the present invention are not limited, but include nucleic acids (including antisense nucleotides of the present invention), peptides, proteins, organic compounds, inorganic compounds, etc. Specifically, it is carried out by bringing these test substances or a sample (test sample) containing them into contact with the cells. Examples of such test samples include, but are not limited to, cell extracts containing test substances, gene library expression products, synthetic low-molecular compounds, synthetic peptides, natural compounds, and the like.
In the screening of the present invention, the conditions for bringing the test substance 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 is forcibly expressed are considered to be master genes involved in the self-renewal ability of ES cells in order to maintain the totipotency of ES cells even in the absence of LIF or feeder cells. It is done. Therefore, the screening method of the present invention includes a method of searching for a substance that controls (activates / suppresses) its activity using the transcription ability of the ECAT4 enhancer / promoter region as an index. By this screening method, a candidate substance effective for maintaining culture of ES cells or a candidate substance effective for differentiating ES cells can be provided. A candidate substance that activates the transcription ability of the ECAT4 enhancer / promoter region is considered useful for maintaining ES cell culture, and a suppressive substance is considered useful for differentiating ES cells.
More specifically, the activity of the ECAT4 enhancer / promoter region in the cells to which the test substance (candidate substance) is added varies (higher / lower) than the level of the cells to which the test substance (candidate substance) is not added. ) If you can choose.
As described above, quantification of the activity of the ECAT4 enhancer / promoter region is carried out using a cell line in which a fusion gene in which a marker gene such as a luciferase gene is linked to the gene region that controls the expression of the ECAT4 gene (expression control region) is introduced. And can be carried out by measuring the activity of the marker gene-derived protein. As a measuring method, for example, Brasier, A. et al. R. (1989) Biotechniques vol. For example, the luciferase activity may be measured by a method according to the description in US Pat.
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, reporter genes such as secretory alkaline phosphatase gene, chloramphenicol acetyltransferase gene, β-glucuronidase gene, β-galactosidase gene, and aequorin gene Can be illustrated.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples, and it is needless to say that ordinary modifications in the technical field of the present invention can be made.

Identification of ECAT4 To identify key transcription factor candidates in pluripotent cells, a digital differential display developed here by National Center for Biological Information was performed. By this method, it is possible to compare the expression frequency of each gene in an expression sequence tag (EST) library derived from various cells and tissues. The EST library (27705 clones) derived from mouse ES cells was compared with the EST library (1328835 clones) derived from various somatic tissues and organs.
(1) Digital differential display In the present invention, using the Digital Differential Display program (http: // www, ncbi.nlm.nih.gov/Unigene/dddd/cgi), cDNA derived from ES cells and other tissues Gene expression in the library was analyzed. ES cell-derived EST libraries were # 274 and 220 (total 27705 entries). The libraries used on behalf of various somatic tissues are # 467, 318, 198, 408, 239, 400, 453, 109, 16, 523, 161, 483, 258, 264, 419, 529, 327, 411, 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 1288835 entries).
As a result of the analysis by the program, 20 genes were found at least 20 times more frequently in ES cells than in somatic tissues. Among these are experimentally identified ES cell-specific marker genes such as Oct3 / 4, UTF1 (Okuda et al., 1998), and Rex1 (Rogers et al., 1991). , Showing the usefulness of this technology. Northern blot analysis confirmed the expression specific to ES cells for at least 9 genes of unknown characteristics. Here, one of them has a homeobox domain as ECAT4. Named (FIG. 1 (A)).
Transcription of ECAT4 was observed in undifferentiated RF8 (Meiner et al., 1996) and MG1.19 (Gassmann et al., 1995) ES cells (FIG. 1 (B)) and was reduced by differentiation induced by retinoic acid. . In 12 organs of adult mice, no ECAT4 transcript could be detected. Furthermore, anti-ECAT4 polyclonal antibody was prepared, and ECAT4 was expressed in four types of ES cell lines of RF8, MG1.19, J1 (Li et al., 1992), and CGR8 (Nichols et al., 1990). Was confirmed (FIG. 1C). When retinoic acid treatment was performed, the expression of ECAT4 was suppressed in all four types of lines. These data indicated that the specific expression of ECAT4, a new homeobox gene, is a characteristic commonly found in ES cells.

A full-length cDNA of ECAT4 consisting of 2184 bases was isolated by ECAT4 protein and a novel homeobox 5′RACE method (FIG. 1 (A)). A single open reading frame encoding a 305 amino acid polypeptide was then identified.
This ECAT4 cDNA has a relatively long 1077 base 3 ′ untranslated region. A homology search revealed that the ECAT4 protein contains a homeobox domain. Phylogenetic analysis showed that this ECAT4 homeobox was closest to the Nk-2 gene family (Harvey, 1996) (FIG. 2 (B)). However, amino acid identity is less than 40%. Furthermore, in ECAT4, valine exists at a position corresponding to tyrosine that is strictly conserved in the NK-2 family. TN domains and NK-2 specific domains that are conserved in the NK-2 family are not present in ECAT4. These data suggest that ECAT4 is not an NK-2 family. ECAT4 has low homology to homeobox domains in other homeobox genes expressed in ES cells such as Oct3 / 4, Pem (Fan et al., 1999) and Ehox (Jackson et al., 2002). I only have sex. There is no known domain in the ECAT4 protein other than the homeobox. ECAT4 is therefore considered to be a new homeobox transcription factor that does not belong to any of the known subfamilies.

Target disruption of mouse ECAT4 gene in mouse ES cells (1) Target disruption of mouse ECAT4 gene Exon 2 of mouse ECAT4 gene having homeobox domain is converted to IRES (internal ribosome binding site) -β-geo (β-galactosidase and neomycin resistance Sex gene fusion) cassette (Mountford et al., 1994) or a targeting vector to replace the hygromycin resistance gene. A 4 kb fragment containing intron 1 and a 1.5 kb 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 targeting vector was introduced into RF8 ES cells by electroporation (Meiner et al., 1996). Screening of genomic DNA for homologous recombination from G418-resistant colonies was performed by Southern blot.
When β-geo target vector was used, 27 out of 96 colonies screened were found to be positive. When the hygr vector was used, 8 out of 27 were positive. Confirmation of homologous recombination was performed using Southern blot (FIG. 2 (B)).
In order to obtain homozygous ES cells, in this study, the hygr vector was introduced into heterozygous mutant cells established using the β-geo vector. Cell sorting was performed with hygromycin on MEF cells using LIF. In 34 out of 83 screened, homologous recombination of the hygr vector was confirmed. Of these, 11 clones were found to be homozygous for the ECAT4 mutation by both Southern blot (FIG. 2 (B)) and PCR (FIG. 2 (C)). Northern blot (Figure 2 (D)) analysis confirmed the lack of ECAT4 expression in homozygous cells. In the other 23 clones, the hygr vector replaced the β-geo cassette.
Next, we attempted to create homozygous cells using the opposite approach. That is, the β-geo vector was introduced into the heterozygous mutant cell established by the hygromycin vector. Cell sorting was performed using G418 on SNL-supporting cells expressing LIF. In 114 of 397 screened, homologous recombination of β-geo vectors was confirmed. Of these, 6 clones were found to be homozygous. The ECAT4-deficient cells obtained by the combination of both behaved 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. Specific amplification using 6047-insS1.2 and 6047-intAS1 primers resulted in a 3.2-kb band from the wild type locus and an 8.5-kb band from the target locus of the β-geo vector. A 4.2-kb band was obtained from the target locus of the hygr vector.
(3) Southern blot analysis concerning screening for homologous recombination Confirmation of homologous recombination was performed using Southern blot analysis. For 5 'Southern blot analysis, genomic DNA was digested with BamHI, separated on a 0.8% agarose gel, and transferred to nylon membranes as previously described (Yamanaka et al., 2000; Yamanaka et al.,). 1998). Hybridization using a 470 bp probe derived from the 5 ′ flanking region resulted in a 8.8 kb band from the wild type locus and a 11.4 kb band from the target locus using the βgeo target vector using the hygromycin vector. A 9.8 kb band was generated from the target locus. Digestion was performed with Scal for 3 ′ Southern blot analysis. Hybridization using a 1 kb probe derived from the 3 ′ flanking region revealed an 11 kb band from the wild type locus, a 15.4 kb band from the target locus using the βgeo target vector, and a target using the hygromycin vector. A 7.3 kb band was generated from the locus.
(4) Identification of mouse genotype After identification of correctly targeted ES cell clones, the genotype of the mouse was determined using 3-primer PCR. The first sense primer 6047-S5 was designed based on exon 2 to amplify the wild type locus. The second sense primer β-geo screening 1 was designed based on the β geo cassette to amplify the targeted locus. A common antisense primer 6047-intAS3 was designed based on intron 2 to amplify both wild type and targeted loci. PCR using these primers resulted in a 850 bp fragment from the wild type locus and a 600 bp fragment from the targeted locus. PCR was performed using ExTaq (Takara) according to the manufacturer's protocol. The PCR program was initial denaturation (94 ° C., 2 minutes), 35 cycles (94 ° C. 30 seconds, 53 ° C. 30 seconds, 68 ° C. 1 minute) and final extension (68 ° C. 7 minutes).

The colonies of ECAT4-deficient cells at the end of selection of differentiation agents for ECAT4 homozygous mutant ES cells into extraembryonic endoderm were morphologically different from those of wild-type and heterozygous ES cells. The central cell appeared undifferentiated, but the surrounding cells were differentiated. When maintained by addition of recombinant LIF on SNL-supporting cells, almost all cells differentiated rapidly and spontaneously into giant circular cells (FIG. 3 (A)). When cultured in the absence of feeder cells, the ECAT4-deficient cells became even more flat and showed a morphology similar to the wall-side endoderm. The growth rate of ECAT4-deficient cells was greatly inhibited (FIG. 3 (B)).
According to Northern blot (FIG. 3 (C)) and RT-PCR (FIG. 3 (D)) analysis, ECAT4 cells are primitive endoderm transcription factors such as GATA4, GATA6, Hinf1β, Hinf4α, Coup-tfI, and Coup-tfII. , Laminin B1, disabled homolog 2 (Dab2), Sparc, tissue plasminogen activator (tPA), mural endoderm markers such as thrombomodulin (TM), and α-fetoprotein (AFP), bone morphogenetic protein 2 (BMP2) ), Visceral endoderm markers such as Indian hedgehog (Ihh) and transthyretin (Ttr) were expressed. However, mesoderm marker T, trophoblast marker Cdx2, primitive ectoderm marker fibroblast growth factor (Fgf) -5, or neuroectodermal marker islet-1 (Isl1) did not increase.
To confirm that ECAT4-deficient cells lost pluripotency, these cells were injected into blastocysts of C57BL / 6 mice. When heterozygous mutant cells were injected, a plurality of chimeric mice were obtained in which ES cells contributed greatly, judging from the fur color. In contrast, when ECAT4 homozygous cells were injected, chimeric mice were not obtained, confirming that the pluripotency of these cells had disappeared.

In order to examine the in vivo function of early embryonic lethal ECAT4 in ECAT4-deficient mice , ECAT4 heterozygous ES cells obtained using the β-geo vector were injected into C57BL / 6 blastocysts. Germline transmission was obtained from three independent ES clones. ECAT4 heterozygous mice were obtained at the expected Mendelian ratio, the overall appearance was normal, and they were able to reproduce. When the embryo before implantation was stained with X-gal, β-geo expression 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 are lethal, heterozygous hybrid embryos at various stages were analyzed here. There were no homozygous embryos at 7.5 dpc and thereafter. Approximately 1/3 of the decidua was empty, indicating that ECAT4-deficient embryos die soon after implantation. In contrast, homozygous and heterozygous blastocysts were found to exist by Mendelian ratio. However, when cultured on gelatin-coated plates, the inner cell mass of ECAT4-deficient blastocysts did not grow (FIG. 4B). These data indicated that ECAT4 is essential for the self-renewal of primitive ectoderm (epiblast).

Efficient establishment of ES cells forcibly expressing ECAT4 (1) Preparation of ES cells forcibly expressing ECAT4 Using the supertransfection system, the coding region of ECAT4 was introduced into the pCAG-IP vector to construct a gene vector . This transgene vector was performed in MG1.19 ES cells using Lipofectamine 2000 (Invitrogen) according to the ES cell protocol provided by the manufacturer. Specifically, MG 1.19 cells were seeded in a 6-well plate (1.2 × 10 ⁵ cells / well), cultured for 24 hours, 8 μg of the transgene was complexed with 4 μl of Lipofectamine 2000, and then the medium. Added to. The next day, the cells were seeded in a 100 mm dish and sorted using 2 μg / ml puromycin. Sorting continued throughout the experiment.
In this transgene vector, a target gene and a puromycin resistance gene are continuously present downstream of a single CAG promoter, and mRNA containing the two genes is transcribed. There is an internal ribosome entry site between them, from which the puromycin resistance gene is translated. Therefore, all cells that have become puromycin resistant will also express the gene of interest. In fact, as a result of introducing ECAT4 into MG1.19 cells and puromycin selection using this method, expression of ECAT4 was observed in 90% or more of the cells. The resulting CAG-ECAT4 cells expressed significantly more ECAT4 transcript (FIG. 5A) and protein (FIG. 5B) compared to control cells transfected with the parent vector.
(2) LIF-independent self-renewal of forcibly expressed ES cells ECAT4 levels in control cells decreased after removal of LIF, but rather increased in CAG-ECAT4 cells. In the absence of LIF, the control cells gradually differentiated and flattened (FIG. 5 (C)), the expression of Oct3 / 4 was suppressed (FIG. 5 (A)), and the cell growth rate was also reduced by the removal of LIF. It decreased (FIG. 5 (D)). On the other hand, CAG-ECAT4 cells maintain an undifferentiated state even after removal of LIF (FIG. 5 (C)), and the expression of Oct3 / 4 also persists (FIG. 5 (A)). Growth caused by LIF removal Resistance to the suppression was also shown (FIG. 5D). From these data, it was shown that the undifferentiated state of ES cells was sufficiently maintained by the constant expression of ECAT4 even in the absence of LIF.
(3) Removal of ECAT4 expression vector and recovery of totipotency pCAG-IP vector is an expression vector maintained extrachromosomally and is known to disappear when culture is continued in the absence of puromycin. Using this, CAG-ECAT4 cells were cultured for 1 month in the absence of puromycin and in the presence of LIF, and the disappearance of the expression of the foreign gene was confirmed by Western blot. The cells differentiated like normal cells when LIF was removed. Moreover, when CAG-ECAT4 cells were microinjected into blast cysts, chimeric mice were obtained. Therefore, even if ECAT4 overexpressing cells were cultured in the absence of LIF, it was confirmed that removal of ECAT4 could regain totipotency. Since CAG-ECAT4 cells sufficiently maintain the undifferentiated state of ES cells even in the absence of LIF, the use of CAG-ECAT4 cells for the production of genetically modified animals makes it easier to produce genetically modified animals. Become.

Identification of DNA recognition sequence using ECAT4 In order to obtain the first clue about transcriptional regulation by ECAT4, here we aimed to determine its DNA recognition sequence by in vitro artificial evolution method (SELEX).
(1) Implementation of SELEX In order to produce a fusion protein consisting of maltose binding protein and ECAT4, the coding region of mouse ECAT gene is introduced into pIH1119 (provided by New England Biolabs), and a transgene vector (pIH1119-ECAT4) is constructed. did. This plasmid pIH1119-ECAT4 was transformed into E. coli. A transformant expressing MBP-ECAT4 was obtained by introduction into E. coli strain BL21AI (Invitrogen).
The cells were cultured in 500 ml of LB medium at 37 ° C. D. After reaching 0.3, IPTG (final concentration 50 μM) was added, followed by culturing at 30 ° C. for 4 hours for protein induction. Cell extracts were collected in 5 ml lysis buffer (20 mM Tris-HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA) containing protease inhibitor cocktail (Nacalai Tesque) and 0.5 mg / ml lysozyme. It was.
The extract was then subjected to 250 μl amylose beads (New England Biolabs) and washed according to the product protocol. SELEX was performed as follows. Double-stranded oligonucleotide synthesis was performed using 1 μg SELEX-N20-Oligo (SEQ ID NO: 11), 1 μg SELEX-N20RV primer (SEQ ID NO: 12), 2.5 μl 10 × Klenow buffer, 4 μl 10 mM dNTPs and 14. The reaction was performed in a reaction solution containing 7 μl of purified water. The mixture was denatured at 95 ° C. for 5 minutes and annealed at 54 ° C. for 5 minutes.
Klenow fragment (0.225 U in 3.6 μl 1 × Klenow buffer) was added to the mixture, which was then incubated at 25 ° C. for 40 minutes. Double-stranded DNA was purified by phenol / chloroform extraction and ethanol precipitation and resuspended in 20 μl of purified water. Performing DNA-protein binding consisted of 20 μl DNA, 30 μl MBP-ECAT4-binding beads, 20 μl 5 × binding buffer (100 mM HEPES-KOH, pH 7.9, 1 mM EDTA, 1 M KCl, and 50% glycerol) and 30 μl In a reaction mixture of water at 4 ° C. for 30 minutes. The beads were washed six times using 100 μl lysis buffer supplemented with 1 mM DTT and 0.2 mM PMSF.
DNA was purified by phenol / chloroform extraction and ethanol precipitation and resuspended in 20 μl of purified water. PCR amplification was performed using 5 μl of the reaction solution. At this time, 5 μl of 10 × ExTaq buffer, 4 μl of 2.5 mM dNTP, 1 μl of 30 μM SELEX-N20FW primer (SEQ ID NO: 13), 1 μl of 30 μM SELEX- N20RV primer, 0.25 μL ExTaq polymerase and 33.75 μL water were included. The amplification product was purified using a Micro-bio spin column (BioRad), concentrated to 20 μl by ethanol precipitation, half of which proceeded to the next round. This procedure was repeated 5 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 the pCR2.1 vector (Invitrogen). Forty clones were randomly selected and sequence analysis was performed.
(2) Sequence analysis As described above, the present inventors have described E.I. E. coli expressed maltose binding protein (MBP) -ECAT4 fusion protein and bound them to amylose resin. A 20-mer random oligonucleotide having adapter sequences at both ends 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 5 times to concentrate 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 and subjected to sequence analysis.
A consensus sequence (G / C) (A / G) (G / C) C (G / C) ATTAN (G / C) was identified by sequence analysis on 37 randomly picked clones. (FIG. 6 (A), SEQ ID NO: 4).
By searching the flanking sequence of the marker gene used in this study, the 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 ), SEQ ID NO: 5). This region is known to be essential for GATA6 expression in the heart (Molkentin et al., 2000; Sun-Wada et al., 2000). Comparison of mouse and human GATA6 gene genomic sequences revealed that this region is highly conserved. The human sequence also contained ECAT4 consensus (FIG. 6 (B), SEQ ID NO: 6).

Identification of ECAT4 Enhancer In order to clarify the molecular mechanism by which mouse ECAT4 gene is specifically expressed in ES cells, an enhancer analysis using a luciferase reporter gene was performed. An approximately 35 kb genomic region containing the mouse ECAT4 gene was divided into 8 fragments shown in FIG. 7 (A) and amplified. This was inserted into the reporter gene shown in FIG. This reporter 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 any transcriptional activity in the differentiated cells, NIH3T3 cells. Therefore, fragment D was considered as an enhancer specific for ES cells.
As a result of the analysis of the ECAT4 enhancer region, a region having high activity specifically for ES cells was present in the region of -6086 / -2292 (fragment D, SEQ ID NO: 7). As shown in FIG. 8 (A), it is active in the region of −4886 / −3444, and among them, the region of −4737 / −4387 is specifically ES cell specific. The highest activity was found. In the region of −4737 / −4387, as shown in FIG. 8 (B), the binding sequence of transcription factor LEF / TCF1, which is downstream of the WNT signal, is present in −4710 / -4703, and this is included. Since the activity decreased when the region of −4737 / −4611 was deleted, this region was considered to be important for the transcriptional activation of ECAT4. In addition, -4524 / -4516 has a binding sequence of STAT3 that is downstream of the LIF signal, and its activity decreased when a point mutation was introduced. Therefore, it was considered to be important for the transcriptional activity of ECAT4. The binding sequence of LEF / TCF1 is also present at −4515 / −4508 immediately after that, suggesting the involvement of ECAT4 in transcriptional regulation. Further, even if the region of −4497 / −4387 was deleted, the activity was reduced, so that this region was considered to be important for the transcriptional activation of ECAT4.

  The present invention provides ES cells in which ECAT4 is forcibly expressed. In addition, the present invention provides ECAT4 consensus sequences, ECAT4 enhancers and their uses. Since the function of ECAT4 as an ES cell self-renewal-promoting transcription factor has been clearly shown, they are extremely useful in the research and development of ES cells.

The base sequence set forth in SEQ ID NO: 4 is a consensus sequence recognized by ECAT4.
The base sequence described in SEQ ID NO: 11 is SELEX-N20-Oligo.
The base sequence described in SEQ ID NO: 12 is a SELEX-N20RV primer.
The base sequence described in SEQ ID NO: 13 is a SELEX-N20FW primer.

Claims (6)

  An ECAT4 enhancer containing the base sequence represented by SEQ ID NO: 15. The base sequence of the ECAT4 enhancer according to claim 1 , comprising a base sequence in which one or more bases are deleted, substituted or added, and has the ability to promote transcription of the ECAT4 gene. 1. The ECAT4 enhancer according to 1 . Claim 1 hybridizes to the DNA under stringent conditions comprising a ECAT4 enhancer nucleotide sequence described, and ECAT4 gene ECAT4 enhancer according to claim 1, characterized in that it has the ability to promote transcription of. Recombinant vector characterized by containing the enhancer according to any one of claims 1 to 3. A transformed cell transformed with the recombinant vector according to claim 4 . Following steps (a), (b) and (c) including, ECAT4 enhancer screening method for a substance having an effect of controlling the activity of:
(A) contacting the transformed cell with a test substance in the enhancer according to any one of claims 1 to 3 was ligated to a reporter gene DNA,
(B) measuring the activity of the reporter gene in the cell contacted with the test substance and comparing the expression level with the activity in the control cell not contacted with the test substance;
Based on the comparison result of (c) above (b), the product of step of selecting a test substance that regulates the activity of ECAT4 enhancer.

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