WO2004067744A1 - Facteur de determination de l'autoreplication de cellules souches embryonnaires - Google Patents

Facteur de determination de l'autoreplication de cellules souches embryonnaires Download PDF

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WO2004067744A1
WO2004067744A1 PCT/JP2004/000790 JP2004000790W WO2004067744A1 WO 2004067744 A1 WO2004067744 A1 WO 2004067744A1 JP 2004000790 W JP2004000790 W JP 2004000790W WO 2004067744 A1 WO2004067744 A1 WO 2004067744A1
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ecat4
seq
cells
dna
gene
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PCT/JP2004/000790
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Japanese (ja)
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Shinya Yamanaka
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Sumitomo Pharmaceuticals Co., Ltd.
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Publication of WO2004067744A1 publication Critical patent/WO2004067744A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a mammalian cell in which ECAT4 is forcibly expressed, an ECAT4 enhancer, a consensus sequence recognized by ECAT4, and uses thereof.
  • Embryonic stem cells derived from the inner cell population of the mammalian blastula, grow rapidly and indefinitely, while retaining the pluripotency, or the ability to differentiate into many types of cells. This property of ES cells is maintained by symmetric self-renewal, which results in two identical stem cell daughter cells during cell division. From these two properties, the potential of a new regenerative medicine that uses ES cells to create tissues and cells and use them for treatment is attracting attention. However, human ES cells also have ethical issues because human embryos must be destroyed for the production of ES cells.
  • the first step toward this goal is the identification of a master gene that functions specifically in pluripotent cells and determines the characteristics of the cells.
  • STAT 3 is activated by leukemia inhibitory factor (LIF), a type of cytokine essential for maintaining pluripotency (Smith et al., (1988). Nature 336, 688-90., Williams et al., (1988). Nature 336, 684-7.). Activated STAT 3 alone supports self-renewal in the absence of LIF (Matsuda et al., (1999). Embo J 18, 4261-9.). Even overnight gene-getting experiments have shown the importance of STAT 3 for self-renewal of ES cells (Raz et al., (1999). Proc Natl Acad Sci USA 96, 2846-51.). However, STAT3 expression is widespread. It is found in a range of cell types and induces differentiation in some cells (Nakajima et al., (1996). Embo J 15, 3651-8.).
  • LIF leukemia inhibitory factor
  • Oct 3/4 Another transcription factor, Oct 3/4, considered to be the key to ES cell self-renewal, is a POU family transcription factor that is expressed in pluripotent cells such as ES cells, ectoderm and primitive embryonic cells I do. Disruption of the mouse Oct 3/4 gene resulted in death of the early embryo shortly after implantation, and the inner cell mass of the blastula lacking Oct 3/4 was not pluripotent and became trophoblast lines. Only differentiate. Niwa eta1 confirmed this in ES cells and showed that suppressing Oct 3/4 resulted in autonomous differentiation into trophoblasts (Niwa et al., (2000). Genet 24, 372-6.). Combining these data with its specific expression pattern suggests that Oct 3/4 may be the master gene for pluripotent cells.
  • An object of the present invention is to provide a cell in which ECAT4 is forcibly expressed, an ECAT4 enhancer, a consensus sequence recognized by ECAT4, and uses thereof.
  • Activating the transcription or function of key self-renewal determinants for ES cells can be expected to improve the efficiency and economy of ES cell culture.
  • it can be expected that undifferentiated cells can be reliably eliminated when ES cells are induced to differentiate into specific functional cells by suppressing the transcription or function of the factor.
  • the risk of appearance of tumor cells (teratomas) derived from keratinocytes can be avoided. Therefore, a major self-renewal determinant is found for ES cells, and an enhancer that regulates the transcription or function of the factor is used to search for a substance capable of inducing or inhibiting the differentiation of ES cells, Very useful for research and development.
  • the present inventors have already found the ECAT4 gene as a gene specifically expressed in ES cells. are doing. Intensive studies of the ECAT4 gene show that targeted disruption of the mouse ECAT4 gene in ES cells results in a spontaneous differentiation that results in an extraembryonic endoderm lineage that expresses high levels of endodermal markers. confirmed. The ECAT4-deficient blastocysts died shortly after implantation, and the inner cell mass of the ECAT4-deficient blastocysts was in vitro and hardly proliferated. We also found that ES cells in which ES cells had the ECAT4 gene forcibly expressed maintain undifferentiated state even when cultured in the absence of LIF, so ECAT4 is a major ES cell. It was found to be an important self-replicating determinant.
  • the inventors have identified a consensus sequence to which ECAT enhancer and ECAT4 bind.
  • the EC AT 4 consensus sequence to which the EC AT 4 protein binds is located 4 kb upstream of the transcription initiation site of the 5′-flanking region of the mouse GAT A 6 gene. It was confirmed that this region was well conserved in the gene sequences of the six genes.
  • the present invention has been completed based on these findings.
  • the gist of the present invention is as follows.
  • Item 1 Self-renewal determinant of embryonic stem (ES) cells consisting of ECAT4,
  • Item 2 The ES cell self-renewal determinant according to Item 1, wherein ECAT4 is mouse ECAT4, human ECAT4 or monkey ECAT4,
  • Item 4 A mammalian stem cell in which ECAT4 is forcibly expressed
  • Item 5 The mammalian stem cell according to Item 4, wherein the stem cell is an ES cell,
  • Item 6 The cell according to Item 4 or 5, wherein ECAT4 is mouse ECAT4, human ECAT4 or monkey ECAT4.
  • Item 7 An amplifier containing a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the polynucleotide represented by SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 or a part thereof Chisense polynucleotide,
  • Item 8 A cell differentiation promoting agent comprising the antisense polynucleotide according to Item 7, Item 9. ECAT4 enhancer,
  • Item 10 The ECAT4 enhancer according to Item 9, comprising a part or all of the nucleotide sequence represented by SEQ ID NO: 7.
  • Item 11 The ECAT4 enhancer according to Item 9, which has the nucleotide sequence of SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.
  • the ECAT 4 enhancer according to Item 11 which is a part of the nucleotide sequence represented by SEQ ID NO: 15, and comprises the nucleotide sequence represented by SEQ ID NO: 17, SEQ ID NO: 8 or SEQ ID NO: 19,
  • the ECAT4 enhancer according to Item 13 which is a part of the nucleotide sequence represented by SEQ ID NO: 14, and contains the nucleotide sequence represented by SEQ ID NO: 15.
  • Item 15 The base sequence of ECAT4 enhancer according to any one of Items 10 to 14, which comprises a base sequence in which one or more bases have been deleted, substituted or added, and the ECAT4 gene ECAT4 sensor according to item 9, which has an ability to promote transcription of
  • Item 17 An EC A4 promoter region comprising the EC AT 4 enhancer according to any one of Items 9 to 16.
  • Item 19 The consensus sequence according to Item 18, wherein the consensus sequence comprises a DNA described in any of the following (a) to (c):
  • Item 20 The consensus sequence according to Item 18, comprising a DNA described in any of the following (a) to (c):
  • Item 21 A recombinant vector characterized by containing the enhancer according to any one of Items 9 to 16, the promoter region according to Item 17, or the consensus sequence according to any one of Items 18 to 20,
  • Item 22 A transformed cell transformed with the recombinant vector according to Item 21,
  • Item 23 A method for screening a substance having an activity of controlling the activity of an ECAT4 enhancer or ECAT4 promoter region, comprising the following steps (a), (b) and (c):
  • Item 24 ES cells transfected with p CAG-IP vector into which ECAT4 gene has been inserted Item 25.
  • a genetically modified animal obtained by injecting the ES cell according to Item 24.
  • FIG. 1 shows the identification of ECAT4.
  • FIG. 2 shows the targeted disruption of the mouse ECAT4 gene.
  • FIG. 3 shows an analysis of ECAT4 deficient ES cells.
  • A Morphology of wild-type RF 8 cells grown on STO feeder cells (top). EC AT 4 cells (middle) grown on STO support cells and EC AT grown without support cells 4 Deficient cells (bottom).
  • B Growth curve.
  • C Northern plot analysis for marker genes.
  • D RT-PCR analysis of the marker gene.
  • Figure 4 shows the analysis of pre-implantation embryos.
  • A Eight-cell embryos and blastocysts obtained from wild-type females and heterozygotes were stained with X-gal.
  • B Blastocysts obtained by heterozygous crossing were grown on gelatin-coated dishes using ES cell culture media. Morphology was recorded 10 days later and genotype was determined by PCR.
  • FIG. 5 shows the forced expression of ECAT4 from the constituent promoters.
  • A PCR-RT analysis showing expression levels of ECAT4, Oct 3Z4 and NAT1 in MG1.19 cells transformed with parental plasmid or CAG-ECAT4. Cells were grown in the presence or absence of LIF.
  • B Western plot confirmed constitutive expression of exogenous ECAT4.
  • C Morphology of control (left) and CAG-ECAT4 expressing cells (right) grown in the presence (top) or absence (bottom) of LIF.
  • D Cell growth rate
  • FIG. 6 shows the transcriptional regulation of ECAT4.
  • FIG. 7 relates to the Enhansa single region analysis of the mouse ECAT4 gene.
  • FIG. 8 relates to the Enhansa single region analysis of the mouse ECAT4 gene.
  • ES cell self-renewal determinant refers to a master gene involved in the self-renewal ability of embryonic stem cells (ES cells) and the like to replicate the same cell without differentiation.
  • the determinant of self-renewal of ES cells can be used as a marker for cells having self-renewal ability such as ES cells. By examining the expression of this factor, it is determined whether the examined cell has self-renewal ability. Alternatively, it can be tested for ES cells. By forcibly expressing this factor, cell proliferation can be safely maintained in the absence of feeder cells and in the absence of cytokines such as IF, while maintaining the totipotency of ES cells. This can be expected to improve the efficiency and economics of ES cell culture. In addition, since there is no risk of contamination with infectious substances such as endogenous virus derived from feeder cells, which is a concern when clinically applied, improved safety can be expected.
  • RNA inhibitors homologous recombination and antisense RNAi (RNA inhibitors) (Experimental method for gene function inhibition by Kazu Makoto Tahira, Yodosha, 2001)
  • RNA inhibitors Experimental method for gene function inhibition by Kazu Makoto Tahira, Yodosha, 2001
  • undifferentiated cells can be expected to be eliminated more reliably than before.
  • safety can be expected to be further improved by avoiding the danger of the emergence of undifferentiated cell-derived tumor cells (teratoma), which is a concern.
  • the “ECAT4” of the present invention is a natural ECAT4 or a recombinant ECAT4, and may be of mammalian origin.
  • mouse ECAT4, rat ECAT4, monkey ECAT4 ⁇ human ECAT4 and the like can be mentioned.
  • the “gene” or “DNA” has a specific nucleotide sequence (SEQ ID NO: 4 to: L 0 14 Not only the ⁇ gene '' or ⁇ MA '' shown in 19), but the biological function is equivalent as long as the function (activity) is the same, or in the case of a gene encoding a protein To the extent possible, it includes “genes” or “DNAs” that encode the congeners (homologs), derivatives and variants thereof. The “gene” or “DNA” encoding these homologs (homologs), derivatives and mutants is specifically shown under any of stringent conditions under any of the aforementioned SEQ ID NOs: 410. "Gene” or “DNA” having a base sequence that hybridizes with a specific base sequence.
  • Stringent conditions include nucleic acid binding to a complex or probe, as described in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). Can be determined based on the melting temperature (Tm). For example, as washing conditions after the hybridization, conditions of about “1XSSC 0.1% SDS 37 ° C.” can be usually mentioned.
  • the complementary strand is preferably one that maintains a hybridized state with the target positive strand even when washed under such conditions.
  • more severe hybridization conditions can be about 0.5XSSC 0.1% SDS 42 ° C, and more severe hybridization conditions can be about 0.1XSSC, 0.1% SDS, 65 ° ().
  • a complementary strand a strand consisting of a base sequence completely complementary to the base sequence of the target positive strand, and 70% or more, preferably 80% or more, More preferably, a chain consisting of a base sequence having homology of 90% or more, more preferably 95% or more can be exemplified.
  • a gene encoding a homologue (homolog) of the specific base sequence (SEQ ID NO: 4 10 14 L9)
  • a gene of another species corresponding to a human gene is Horn oloGene (http :, ncbi. It can be identified by nlm. nih. gov / HomoloGene /).
  • the specific human nucleotide sequence is subjected to BLAST (Proc. Natl. Acad. Sci. USA., 90: 5873-5877, 1993 http://ww.ncbi.nlm.nih.gov/BLAST/) to be matched.
  • the gene or DNA does not matter whether it is a functional region, and may include, for example, an expression control region, a coding region, an exon or an intron.
  • the EC AT 4 gene (DNA) represented by the specific nucleotide sequence (SEQ ID NO: 8-10) are used for the purpose of including genes (DNA) encoding their homologs, mutants and derivatives.
  • the human ECAT4 gene described in SEQ ID NO: 9 GenBank Accession NO. AB093576
  • the mouse ECAT4 gene described in SEQ ID NO: 8 GenBank Accession NO. AB093574
  • the SEQ ID NO: 10 The described monkey EC AT4 gene and a rat homolog are included.
  • RNA Ribonucleic acid
  • DNA DNA
  • the above-mentioned RNA includes total fragile, mRNA and synthetic RNA.
  • protein or “(poly) peptide” includes not only “protein” or “(poly) peptide” represented by a specific amino acid sequence (SEQ ID NOs: 1 to 3), but also Fragments, homologs (homologous splice variants), variants, derivatives, amino acid modifications, etc., are included as long as their biological functions are equivalent.
  • homologues include proteins of other species such as mouse and rat corresponding to human proteins, and these are obtained by 11011101 ( ⁇ 611601 0: ⁇ ⁇ .1 ⁇ bi.nlm.nih.gov/HomoloGene/). It can be determined a priori from the nucleotide sequence of the identified gene.
  • Variants also include naturally occurring allelic variants, non-naturally occurring variants, and variants having an amino acid sequence altered by artificial deletion, substitution, addition and insertion. You.
  • examples of the mutant include those that are at least 70%, preferably 80%, more preferably 95%, and even more preferably 97% homologous to the protein or (poly) peptide having no mutation.
  • Amino acid modifications include naturally occurring amino acids and non-naturally occurring amino acids, and specifically include phosphorylated amino acids. Therefore, when the term “ECAT4 protein” or simply “ECAT4” is used herein, unless otherwise specified, the specific amino acid sequence (SEQ ID NO: 1) To 3), and their congeners, variants, derivatives, amino acid modifications and the like. Specifically, human ECAT 4 having the amino acid sequence of SEQ ID NO: 2, mouse ECAT 4 having the amino acid sequence of SEQ ID NO: 1, and amino acid sequence of SEQ ID NO: 3 Monkey ECAT4, and their rat homologs.
  • the “mammalian stem cell in which ECAT4 is forcibly expressed” of the present invention is a cell obtained by forcibly expressing ECAT4 in a mammalian cell.
  • Mammalian cells are cells derived from tissues, organs, etc. of mammals such as humans, monkeys, mice, rats, etc., and may be primary cells taken from individuals, cultured cells, or mammalian cells. Any animal-derived cells may be used.
  • Mammalian cells include, for example, commercially available cultured cells (such as ATCC), mouse ES cells, monkey ES cells ⁇ human ES cells, and more preferably, mouse ES cells, monkeys that differentiate in the absence of IF.
  • ES cells ⁇ embryonic stem cells such as human ES cells, but are not limited thereto.
  • an expression vector containing the ECAT4 gene may be introduced into cells.
  • Specific methods for introducing an expression vector include the calcium phosphate method, DEAE-dextran, and the like. And known methods such as electroporation, a method using a lipid for transfection (Lipofectamine, Lipofectin; Gibco-BRL), and a microinjection method.
  • An expression vector containing the ECAT4 gene can be produced based on ordinary genetic engineering techniques.
  • the expression vector used here can be appropriately selected depending on the host used, the purpose, and the like, and examples thereof include a plasmid, a phage vector, and a virus vector.
  • plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.K pRc / RSV and pRc / CMV, and viral vectors such as retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors.
  • the vector may optionally have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, and a terminator. Further, it may have a gene encoding a fusion protein of the marker overnight protein and ECAT4.
  • the “ma-chi-ichi-gene” refers to a gene whose phenotype is clear and is useful for genetic analysis, and the expression of which can be directly observed when the gene is introduced.
  • Examples include fluorescent protein genes such as CFP and YFP, which are mutants of GFP and GFP, but are not limited thereto. All that can confirm that the gene has been introduced in the above are included in the category of the marker-gene of the present invention.
  • a protein is a protein encoded by a marker gene.
  • ECAT 4 By introducing a vector to which such a marker gene is added, cells expressing ECAT 4 can be easily detected. Furthermore, by using ES cells into which such a gene has been introduced, only stem cells that express ECAT4 can be selected. Thus, when ES cells are divided into functional cells, undifferentiated cells and differentiated cells can be selected. It can also be used as a marker that can select This can avoid the danger of transplanting undifferentiated cells into a living body, which may be at risk of teratoma formation. Furthermore, an expression vector containing the ECAT4 gene can also be produced using a conditional gene expression control system.
  • a forced expression system in which the introduced gene of interest is expressed in the presence of tetracycline but not in the absence of tetracycline can be mentioned (Niwa et al., (2000). Nat Genet 24, 372-6.).
  • Cells into which these vectors have been introduced can switch on and off ECAT4 expressing cells depending on the culture conditions, so that ECAT4 is stably forcibly expressed between ES cells, and expression is induced when differentiation is induced. It is thought that differentiation can be efficiently induced by shutting off, and can be used for various purposes.
  • the expression vector containing the ECAT4 gene may be a vector having the property of disappearing under certain conditions. Specifically, for example, a pCAG-IP vector and the like can be mentioned. ES cells into which the ECAT4 expression vector having such properties has been introduced are very useful for producing genetically modified animals such as transgenic mice. In the ES cells into which the vector has been introduced, the vector itself disappears from the cell under certain conditions. Genetically modified animals can be prepared more simply and reliably than controlling the expression level of ECAT4.
  • CAG-EC AT cells produced using a pCAG-IP vector containing the ECAT 4 gene can be cultured in the absence of LIF ⁇ feeder cells.
  • the operation of introducing another vector for expressing, knocking out, knocking down or knocking in the desired target gene into the target gene is very easy.
  • the genetically modified animal is an animal in which the standard gene is overexpressed, knocked out, knocked down or knocked in.
  • the genetically modified animal can be prepared by a known method, and the animal may be a mammal, and examples thereof include a mouse and a rat. ,
  • the antisense polynucleotide of the present invention has a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the ECAT4 gene or a part thereof, and can suppress the expression of the ECAT4 gene. Any substance having an action may be used, and examples thereof include antisense RNA and antisense DNA.
  • the substantially complementary nucleotide sequence is, for example, about 70% or more, preferably about 80% or more, more preferably about 90% or more of the nucleotide sequence complementary to the nucleotide sequence of the ECAT4 gene. % Or more, most preferably about 95% or more homology.
  • about 70% or more, preferably about 80% or more, of the complementary sequence of the base sequence (for example, the base sequence near the start codon) of the portion encoding the N-terminal portion of the base sequence of the ECAT4 gene More preferably, an antisense polynucleotide having about 90% or more, most preferably about 95% or more homology is suitable.
  • a polynucleotide having a complementary base sequence refers to the full-length sequence of a polynucleotide consisting of the base sequence shown in each of the above-mentioned sequence numbers, or at least 15 continuous bases in the base sequence.
  • base pairing such as A: T and G: C 1.4 means polynucleotides that are in a complementary relationship with each other.
  • a complementary strand is not limited to the case where it forms a completely complementary sequence with the base sequence of the target positive strand, but has a complementary relationship sufficient to hybridize with the target positive strand under stringent conditions. May be provided.
  • polynucleotides on the positive chain side include not only those having the base sequence of the ECAT4 gene or a partial sequence thereof, but also those having a more complementary relationship to the base sequence of the complementary chain. Chains can be included.
  • the antisense polynucleotide is usually composed of about 10 to 1000 bases, preferably about 15 to 500 bases, and more preferably about 16 to 30 bases.
  • the phosphate residues (phosphates) of each nucleotide constituting antisense DNA are chemically modified with, for example, phosphorothioate, methylphosphonate, phosphorodithionate, etc. It may be substituted with a phosphate residue.
  • These antisense polynucleotides can be produced using a known DNA synthesizer or the like.
  • Such antisense polynucleotides can hybridize to the RNA of the EC AT4 gene and can inhibit the synthesis or function of the RNA, or can interact with the RNA via the RNA. It can regulate and control the expression of AT4 gene.
  • the antisense polynucleotide of the present invention is useful for regulating and controlling the expression of ECAT4 in vivo and in vitro, and is useful for reliably eliminating undifferentiated cells as described above. is there. 5 'end hairpin loop of protein gene, 5' end 6—base pair repeat, 5 'end untranslated region, polypeptide translation initiation codon, protein coding region, ORF translation stop codon, 3' end untranslated region, 3 '
  • the terminal palindrome region or the 3 ′ terminal hairpin loop can be selected as a preferable target region, but any region in the protein gene can be selected as the target.
  • Antisense polynucleotides can be polynucleotides containing D-reports, polynucleotides containing D-reports, N-glycosides of purine or pyrimidine bases, or other types of polynucleotides.
  • nucleotide backbone eg, commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers
  • other polymers containing special bonds e.g., such polymers are found in DNA and RNA
  • nucleotides having a configuration that allows base pairing and base attachment may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, and may further comprise unmodified polynucleotides (or unmodified oligonucleotides). ), With known modifications, e.g., labeled, capped, methylated, or substituted with one or more natural nucleotides, as known in the art.
  • an intramolecular nucleotide for example, having an uncharged bond (eg, methylphosphonate, phosphotriester, phosphoramide, oleambamate, etc.), a charged bond or a sulfur-containing bond (eg, phosphorothioate)
  • proteins eg, nucleases, nuclease inhibitors, toxins, antibodies, Compounds having side-chain groups such as guanyl peptides, poly-L-lysine, etc., sugars (eg, monosaccharides), compounds having interactive compounds (eg, acridine, psoralen, etc.), chelating compounds ( For example, those containing metals, radioactive metals, boron, oxidizing metals, etc., those containing alkylating agents, and those with modified bonds (eg, alpha-anomeric nucleic acids, etc.).
  • nucleoside may include not only those containing purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleotides and modified nucleotides may also be modified at the sugar moiety, e.g., where one or more hydroxyl groups have been replaced with octalogens and aliphatic groups, or functional groups such as ethers, amines, etc.
  • the antisense polynucleotide of the present invention may comprise RNA, DNA or modified nucleic acid (RNA , DNA).
  • modified nucleic acid include a sulfur derivative of a nucleic acid, a thiophosphate derivative, a polynucleoside amide / a nucleic acid that is resistant to degradation of oligonucleoside amide, and the like.
  • the antisense polynucleotide of the present invention can be designed, for example, as follows. In other words, it makes the antisense polynucleotide more stable in the cell, enhances the cell permeability of the antisense polynucleotide, increases the affinity for the target sense strand, and In some cases, the toxicity of the antisense polynucleotide is reduced. Many such modifications have been reported, for example, in Pharm Tech Japan, vol. 8, p. 247 or p. 395, 1992, Antisense Research and Appli- cations, CRC Press, 1993.
  • the antisense polynucleotide of the present invention may be provided in a special form such as ribosome or microsphere, or may be provided in an added form more suitable for gene therapy.
  • additional forms include polycations, such as polylysine, which act to neutralize the charge of the phosphate backbone, and lipids, which increase the interaction with cell membranes and increase the uptake of nucleic acids. (Eg, phospholipids, cholesterol, etc.).
  • Preferred lipids for addition include cholesterol and its derivatives (eg, cholesteryl chromate formate, cholic acid, etc.).
  • nucleic acid can be attached via a base, sugar, or intramolecular nucleoside linkage.
  • Other groups include capping groups specifically located at the 3 'or 5' end of nucleic acids that prevent degradation by nucleases such as exonucleases and RNases. No. Such capping groups include, but are not limited to, hydroxyl-protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the inhibitory activity of an antisense polynucleotide can be examined using the screening method of the present invention.
  • the antisense polynucleotide of the present invention may be double-stranded, and binds to RNA encoding ECAT4 and destroys the RNA or suppresses its function. That is, a part of the RNA encoding ECAT4 and its complementary Double-stranded RNAs containing various RNAs are also included in the antisense polynucleotide of the present invention.
  • the use of the antisense polynucleotide is similar to that of ordinary gene therapy.
  • an antisense oligonucleotide or a chemically modified product thereof is directly administered to a target cultured cell or tissue. This can be performed by a method for controlling the expression of the target gene.
  • the antisense polynucleotide of the present invention can be administered to cells as it is or by incorporating it into a vector for gene therapy. Also in these cases, the dose and the administration method vary depending on the type of cells, the number of cells, and the like, and those skilled in the art can appropriately select them.
  • the antisense polynucleotide or a chemically modified product thereof can bind to the sense strand mRNA in cells and regulate the expression of the target gene, that is, the expression of ECAT4, and thus the function (activity) of ECAT4. Can be controlled.
  • the antisense polynucleotide or the chemically modified product used is preferably 10 to 1000 bases, more preferably 15 to 500 bases, and most preferably Preferably, it should have a length of 16 to 30 bases.
  • the antisense oligonucleotide or a chemically modified product thereof can be formulated (reagentized) using a commonly used stabilizer, buffer, solvent or the like.
  • the method using the above-mentioned recombinant virus includes, for example, the virus genome of the present invention such as retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus, vaccinia virus, polio virus, and symbis virus.
  • a method of incorporating an antisense polynucleotide into a living body and introducing it into a living body Among them, a method using a retrovirus, an adenovirus, an adeno-associated virus or the like is particularly preferable.
  • the non-viral introduction method include a ribosome method and a lipofectin method, and a liposome method is particularly preferable.
  • Other non-viral introduction methods include, for example, a microinjection method, a calcium phosphate method, and an electroporation method.
  • the cell differentiation promoting agent of the present invention includes the above-mentioned antisense polynucleotide or a chemically modified product thereof, a recombinant virus containing the same, and an infection into which these viruses have been introduced.
  • a cell or the like is used as an active ingredient.
  • the agent for promoting cell differentiation of the present invention can be used as a reagent or a medicine.
  • the administration form and the like of the composition can be appropriately determined depending on the target cell or tissue.
  • the medicament or reagent of the present invention may be, for example, a form in which a virus vector containing the antisense polynucleotide of the present invention is embedded in ribosomes or membrane fusion liposomes (eg, Sendai virus (HVJ) -ribosome).
  • ribosomes or membrane fusion liposomes eg, Sendai virus (HVJ) -ribosome.
  • ribosome formulations include suspensions, cryogens, and centrifuged concentrated cryogens.
  • the vector containing the antisense polynucleotide of the present invention may be in the form of a cell culture solution infected with the introduced virus.
  • the dosage of the active ingredient in these various forms of preparation can be appropriately adjusted depending on the purpose.
  • an antisense polynucleotide against the ECAT4 gene about 0.001 x -lOOmg, preferably about 0.001 g-lOmg may be added / administered once every several days to several months.
  • 0.001 x -lOOmg preferably about 0.001 g-lOmg
  • 0.0001 ag to 1 OOfflg preferably 0.001 ag to 1 Omg of the antisense nucleotide of the present invention may be added once every few days to several months.
  • the present invention provides (i) a double-stranded RNA containing a part of the RNA encoding ECAT4 and its complementary RNA, (ii) a medicine containing the double-stranded RNA, (iii) an EC A lipozyme containing a part of RNA encoding AT4, (iv) a medicine containing the lipozyme, (V) an expression vector containing a gene (DNA) encoding the lipozyme, and the like are also provided.
  • antisense polynucleotide, double-stranded RNA, lipozyme and the like can also break down RNAs transcribed from the DNA of the present invention or inhibit its function. It can be used as a reagent or therapeutic agent such as double-stranded RNA, lipozyme which can suppress the function of ECAT4 or the DNA encoding it.
  • Double-stranded RNA can be designed and manufactured based on a polynucleotide sequence encoding ECAT4 according to a known method (eg, Nature, 411, 494, 2001).
  • the lipozyme can be designed and manufactured based on the sequence of the polynucleotide encoding ECAT4 according to a known method (eg, TRENDS in Molecular Medicine, Vol. 7, pp. 221, 2001). For example, it can be produced by substituting a part of a known lipozyme sequence with a part of RNA encoding ECAT4.
  • RNA encoding T4 As a part of the RNA encoding T4, a sequence near a consensus sequence NUX (where N indicates all bases and X indicates a base other than G) which can be cleaved by a known lipozyme, and the like can be mentioned.
  • NUX consensus sequence
  • the above-mentioned double-stranded RNA or lipozyme is used as the above-mentioned reagent or medicine, it can be prepared and administered in the same manner as the antisense polynucleotide.
  • the above-mentioned expression vector is used in the same manner as a known gene therapy method and the like, and is used as the above reagent or medicine.
  • ECAT4 enhancer ECAT4 promoter region, consensus sequence recognized by ECAT4, recombinant vector and transformed cell of the present invention
  • ECAT4 enhancer refers to a base sequence that promotes the transcription of ECAT4.
  • Use of the ECAT4 promoter region including the ECAT4 enhancer of the present invention enables efficient transcription of the ECAT4 gene and the like.
  • the ECAT4 enhancer of the present invention comprises (i) an ECAT4 enhancer comprising a part or all of the nucleotide sequence represented by SEQ ID NO: 7, (ii) SEQ ID NO: 17, SEQ ID NO: 18 or a sequence thereof.
  • ECAT 4 (Vii) a DNA comprising the nucleotide sequence of ECAT4 enhancer described in any one of the above (to (v) and a stringent, which has the ability to promote transcription of offspring.
  • An EC AT4 enhancer characterized in that it hybridizes under various conditions and has the ability to promote the transcription of the ECAT4 gene.
  • the DN of the region from position -4737 to position -4611 of the nucleotide sequence shown in FIG. 8 ( ⁇ ) (the region from position 1 to position 127 in SEQ ID NO: 15) ECAT4 enhancer containing ⁇ , and the region from position -4497 to position -4387 of the nucleotide sequence shown in FIG. 8 (B) (the region from position 241 to position 351 in SEQ ID NO: 15) ) DNA-containing EC AT4 enhancer.
  • the “promoter region” is a type of regulatory gene and is a region on DNA that determines the transcription initiation site of a gene and directly regulates its frequency. To induce promoter elements under cell-type or tissue-specific regulation, or promoter-dependent gene expression induced by exogenous signals or factors (eg, transcriptionally activated proteins) It may contain sufficient promotion elements.
  • the “ECAT4 promoter region” of the present invention contains the aforementioned ECAT4 enhancer of the present invention, and may be any DNA as long as it is a DNA having the promoter activity of ECAT4. .
  • DNA consisting of a part or all of the base sequence shown in SEQ ID NO: 14 and the like can be mentioned.
  • the “consensus sequence recognized by ECAT4” of the present invention is a gene sequence of a site recognized by ECAT4 when ECAT4 binds to a gene and exhibits a transcription control action.
  • the consensus sequence recognized by ECAT4 of the present invention includes:
  • one or more bases is preferably one to three bases, and more preferably one to two bases.
  • the “part of the sequence” in (iii) may be, for example, the DNA of 5 to 10 nucleotides from the side end of the nucleotide sequence represented by SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 16, or 3 'DNA from several bases to 10 bases from the side end can be mentioned, but is not limited thereto.
  • the ECAT4 enhancer of the present invention, the consensus sequence recognized by ECAT4, and the ECAT4 promoter region are human or other mammalian cells (for example, human lymphocyte cells, mouse fibroblasts, mouse ES cells, It may be any of genomic DNA, cDNA and synthetic DNA derived from rat ES cells, human ES cells) or any tissue where such cells exist.
  • Examples of the method for preparing the ECAT4 enhancer, the consensus sequence recognized by ECAT4, and the ECAT4 promoter region of the present invention include chemical synthesis, PCR, and hybridization.
  • an automatic DNA synthesizer for example, a DNA synthesizer model 38OA (manufactured by ABI) can be used.
  • a genomic library of type ⁇ ⁇ ⁇ is described, for example, in "Mocular C 1 oning: AL abo rato ry Manua 1 2nd edition” (1989), Cold Spring Harbor Labo ratory Press, etc. It can be prepared from tissues of mammals such as humans and mice according to the methods described. In addition, commercially available genomic DNA such as human genomic DNA (manufactured by Clonetech) and commercially available genomic libraries such as human genome walker kit (manufactured by Clonetech) can be used. Next Then, perform PCR using primers corresponding to the promoter to be amplified.
  • the primer can be appropriately designed based on the nucleotide sequence represented by SEQ ID NO: 7, SEQ ID NO: 14 or SEQ ID NO: 15, and a restriction enzyme recognition sequence or the like is added to the 5 ′ end thereof. Is also good.
  • the DNA amplified as described above is described in ⁇ Mo ecu 1 ar C 1 oning: AL abo rato ry Manu al 2nd edition '' (1989), Cold Spring Har Harbor rboratory Press, Cu rr ent Protocols In Mo 1 ecu 1 ar Biol ogy ”(1987), John Wiley & Sons, Inc. ISBN 0—471—50338—X It can be cloned into a vector according to the method.
  • cloning can be performed using a plasmid vector included in a TA cloning kit manufactured by Invitrogen or a plasmid vector such as pB1uescript II manufactured by Stratagene.
  • the nucleotide sequence of the cloned DNA is described in F. Sanger, S. Nickl en, AR Cou ls on, Proceedings of Na ti on al Ac ad emy of S ci ence USA (1977), 74, 5463_
  • the analysis can be carried out by the didoxita-mining method described in 5467 and the like.
  • the DNA used for the probe may be a DNA having at least a part of a base sequence such as an enhancer to be prepared or a promoter region, for example, a base sequence represented by any one of SEQ ID NOs: 4 to 7 and 14 to 19 or DNA comprising a continuous partial base sequence and having a chain length of 16 bases or more and 160 bases or less, wherein one or more bases are deleted, substituted or added in the base sequence of the DNA And DNA that hybridizes with the DNA under stringent conditions.
  • a base sequence such as an enhancer to be prepared or a promoter region
  • the DNA used for the probe can be obtained, for example, by the above-mentioned usual DNA preparation method such as a chemical synthesis method, a PCR, and a hybridization method.
  • the DNA used for the probe is a gene that encodes ECAT4 by itself. It may have the ability to control the photo.
  • a radioisotope for example, Random Lab elling Kit manufactured by Ba Ringer and Takara Shuzo can be used, and dCTP in the ordinary PCR reaction composition can be used as [a - 32 P] instead of dCTP, by performing to PC R reacting the DN a to ⁇ used to probe, it is also possible to perform labeling.
  • a fluorescent dye for example, ECL Direct Nucleic Acid Label and D itection on Syst em mersh am Pharmacia Biotech
  • a DNA library for hybridizing probes for example, a genomic DNA library derived from animals such as rodents such as mice can be used.
  • genomic DNA library a commercially available genomic DNA library can be used, and “Moecular C 1 oning: AL aboratory Manu al 2nd edition” (1989), Cold Spring Harr Borr Laboratory Press, "CurrentProtocols In Molecular BiologyJ (1987), John Wiley & Sons, Inc. IS BN 0—471—50338_X, etc.
  • a ⁇ vector such as ⁇ FIXII, ⁇ EMBL 3, ⁇ EMBL4, ⁇ DASH II manufactured by Stratage, Gig ap ackpack
  • a genomic DNA library can be prepared using in vitro packaging (Stratagene) or the like and used.
  • Examples of the hybridization method include colony hybridization and plaque hybridization, and it is preferable to select a method according to the type of the vector used for preparing the library.
  • the library used is a library constructed with a plasmid vector
  • colony hybridization can be performed. Specifically, first, transformants are obtained by introducing the library DNA into a host microorganism, and the obtained transformants are diluted, plated on an agar medium, and cultured at 37 until colonies appear. Also used la If the library is a library constructed with a phage vector, black hybridization can be performed.
  • the host microorganism and the phage of the library are mixed under conditions that allow infection, and then mixed with a soft agar medium, which is then spread on an agar medium. Incubate at 37 ° C until plaques appear. More specifically, for example, Mo 1 ecu 1 ar Cl on i ng 2 nd edit on (J. Sam Rok, EF Frisch, T. Maniatis, Cold Spring Har bor L abo rato ry P ress 1989 years) 2. according to the method described in 2.65, etc. 60, a density of 0. 1 to 1. O pfu agar lmm 2 per a NZY agar medium, about 9. 0 X 10 5 pfu file spread one di libraries, cultured 6-10 hours at 37 ° C.
  • a membrane filter is placed on the surface of the agar medium on which the above-mentioned culture has been carried out, and the transformed cells or phage carrying the plasmid are placed in the membrane. Transfer to a plain filter.
  • This membrane filter is subjected to an alkali treatment, a neutralization treatment, and a treatment for fixing DNA to the filter. More specifically, for example, in the case of plaque hybridization, the cloning and sequence are carried out according to the usual method described in a manual for plant biotechnology experiments (edited by Watanabe and Sugiura, Rural Bunkasha 1989) and the like.
  • a dinitrocellulose filter or a nylon filter such as Hybond- ⁇ '(Amersham Pharmacia Biotech)
  • Hybond- ⁇ ' Amersham Pharmacia Biotech
  • the filter was immersed in an alkaline solution (1.5 M sodium chloride, 0.5 N sodium hydroxide) for about 3 minutes to dissolve the phage particles and elute the phage DNA over the filter.
  • an alkaline solution 1.5 M sodium chloride, 0.5 N sodium hydroxide
  • a neutralization solution 1.5 M sodium chloride 0.5 M Tris-HCl, H7.5
  • the phage is removed. Immobilize the DNA on the filter. Hybridization is performed using the filter thus prepared and the probe. Reagents and temperature conditions for performing hybridization are described in, for example, “DNA cl on in DMG 1 o Ver”.
  • the senor contains 450-90 OmM sodium chloride, 45-9 OmM sodium citrate, contains sodium dodecyl sulfate (SDS) at a concentration of 0.1-1.0% (W / V), Prehybridization containing specific DNA at a concentration of 0 to 200 gZmL and optionally containing albumin, ficoll, polyvinylpyrrolidone, etc. at a concentration of 0 to 0.2% (W / V).
  • SDS sodium dodecyl sulfate
  • Chillon solution preferably prehybridization containing 90 OmM sodium chloride, 9 OmM sodium citrate, 1% (W / V) SDS and 100 gZmL denatured Ca1f-thymus DNA
  • the Zeshiyon solution prepared in the ratio of the filter one 1 cm 2 per 50 to 200 were prepared as L, 1 to 4 hours at 42 to 68 ° C by immersing the filter one to the solution, preferably Incubate at 45 ° C for 2 hours.
  • a clone having the DNA can be isolated.
  • the filter is exposed to an imaging plate (Fuji Film) for 4 hours, and then the plate is analyzed using BAS 2000 (Fuji Film) to detect a signal.
  • the obtained phage particle eluate was subjected to Molecular Cloning 2ndedition (J. Sam Rook, EF Frisch, T. Maniatis, Cold Sp rig Har Har bo r Labo rato ry
  • a phage clone containing a DNA consisting of a base sequence that hybridizes with the probe used can be obtained. Screening by hybridization as described above
  • the DNA contained in the clone obtained as described above is subcloned into a plasmid vector, which is easy to prepare and analyze, such as commercially available pUC18, pUC19, pBLUESCR I PT KS +, pBLUESCR I PT KS-, etc. DNA was prepared and prepared by F. Sanger, S.
  • the nucleotide sequence can be determined using the didoxinating method described in, for example.
  • the preparation of the sample used for the nucleotide sequence analysis is performed, for example, by Molecular Cloning 2nd edition (J. Sam Rook, EF Frisch, T. Maniatis, Cold Spring Harbor L aboratory Press, 1989) 13. It can be carried out according to the primer extension method described in 15 and the like.
  • the phage clone was prepared using the Molecular Cloning 2nd edition (J. Sambrook, EF Frisch, T. Maniatis, Cold Spring Laboratory Press, 1989).
  • the ability to control transcription means, for example, an activity of initiating and / or promoting transcription of a gene located downstream of a promoter region (hereinafter, also referred to as promoter activity). ).
  • the ECAT4 enhancer of the present invention, the consensus sequence recognized by ECAT4, and the ECAT4 promoter region have mutations in the sequence containing the base sequence shown in any one of SEQ ID NOs: 4 to 7 and 14 to 19. It may be made by introducing. Specifically, for example, the method described in A. Greener, M. Callahan, Strategies (1994) 7, 32-34, etc. is used at random. Can be obtained by introducing a mutation, such as W. Kramer, eta 1., Nucleic Acids Research (1984) 12, 9441, or W. Kramer, HJ Frits, Method sin En.
  • a chimeric DNA is prepared in which one or several partial nucleotide sequences of the EC AT4 promoter region including the nucleotide sequence shown in SEQ ID NO: 15 are replaced with a part of the DNA of another promoter. For example, S. Hen i ko ff, eta 1., Gene (1984) 28, 351, C. Yan isch—Pe rr on, eta 1., Gene (1985) ) The method described in 33, 103 etc. can be used.
  • the promoter region of the present invention prepared by the above-described various methods can be prepared by a conventional method, for example, “Tamura Takaaki (published by Yodosha), New Transcription Control Mechanism (2000)”, pp. 33-40, “Nomura According to the method described in Shintaro and Toshiki Watanabe supervised by Shujunsha (published by Shujunsha Publishing Co., Ltd., Deisotope Experiment Protocol (1998)), a part of the base is deleted while maintaining the promoter activity. (That is, prepared by excising with an appropriate restriction enzyme) can also be used as the promoter region of the present invention.
  • the ability of the obtained DNA to regulate the transcription of the ECAT4 gene can be confirmed by the method described below.
  • ECAT 4 enhancer or ECAT 4 promoter region of the present invention (hereinafter, referred to as “E CAT 4 enhancer / promo overnight region” in the present specification) is inserted.
  • the recombination vector may be any recombination vector that can function in a desired cell.
  • a known suitable promoter can be operably linked.
  • the recombinant vector of the present invention selects cells into which the recombinant vector has been introduced. For example, a kanamycin resistance gene, an octaidaromycin resistance gene, a neomycin resistance gene, and the like.
  • a position where the enhancer-promoter region of the present invention and a desired gene are operably linked on a recombinant vector, for example, the enhancer-Z promoter region.
  • a gene insertion site is further provided downstream of the site into which the gene is inserted, it can be preferably used for construction of a recombinant vector for expressing a desired gene in cells.
  • the gene insertion site is, for example, a nucleotide sequence that can be specifically recognized and cleaved by a restriction enzyme usually used in genetic engineering techniques, and is present only on a recombinant vector having a region of the enhancer / promoter of the present invention. Preferred types of restriction enzyme recognition sequences are preferred.
  • the recombinant vector examples include a pUC plasmid (pUC118, pUC119 (manufactured by Takara Shuzo) and the like), a pSC101 plasmid, and a pBR322 plasmid (manufactured by Boehringer Mannheim).
  • pUC118, pUC119 manufactured by Takara Shuzo
  • pSC101 plasmid a pBR322 plasmid
  • Boehringer Mannheim pBR322 plasmid
  • a marker gene for selecting cells into which the recombinant vector has been introduced for example, a kanamycin resistance gene, a hygromycin resistance gene, a neomycin resistance gene, etc.
  • a marker gene for selecting cells into which the recombinant vector has been introduced for example, a kanamycin resistance gene, a hygromycin resistance gene, a neomycin resistance gene, etc.
  • a position where the consensus sequence of the present invention and a desired gene are operably linked on the recombination vector, for example, downstream of a site where the consensus sequence is inserted.
  • a suitable promoter and gene insertion site are further provided, it can be preferably used for construction of a recombinant vector for expressing a desired gene in cells.
  • the gene insertion site is, for example, a nucleotide sequence that can be specifically recognized and cleaved by a restriction enzyme usually used in genetic engineering techniques, and is a recombinant having a consensus sequence recognized by ECAT4 of the present invention. Restriction enzyme recognition sequences of the type present solely on the vector are preferred.
  • Specific examples of the recombinant vector include a pUC plasmid (pUC118, pUC119 (Takara Shuzo) etc.), a pSC101 plasmid, and a pBR322 plasmid (Boehringer Mannheim). Be mentioned
  • E CAT 4 enhancer / promoter In order to examine the binding of the consensus sequence recognized by ECAT4, a detectable structural gene may be ligated downstream of the ECAT4 enhancer / promoter region or the consensus sequence recognized by ECAT4.
  • Various repo overnight genes are used as structural genes linked downstream of the enhancer / promoter region or consensus sequence. As the repo overnight gene, luciferase gene, CAT (Chloramphenicol acetyl transferase) gene, alkaline phosphatase gene, and] -galactosidase gene are widely used.
  • any other structural gene can be used as long as there is a method for detecting its gene product.
  • the above-mentioned structural gene can be incorporated into a vector by recognizing the ECAT4 enhancer / promoter region or ECAT4.
  • the above-mentioned structural gene may be ligated to an appropriate restriction enzyme cleavage site located downstream of the consensus sequence, for example, a commercially available vector such as pGL3 Basic, pGL3 promoter (Promega). It can also be used.
  • Escherichia bacteria for example, Escherichia bacteria, Bacillus bacteria, yeast, insect cells, animal cells, and the like are used.
  • animal cells include monkey cell COS-7, Vero, Chinese Hams Yuichi cell CHO (hereinafter abbreviated as CHO cell), dh fr gene-deficient Chinese hamster cell CH O (hereinafter CH ⁇ (dhfr_) cell) ), Mouse L cells, mouse AtT—20, mouse myeloid cells, rat GH3, mouse fibroblasts 3T3—L1, human liver cancer cells HepG2 (hereinafter referred to as HepG2 cells).
  • CHO cell monkey cell COS-7
  • Vero Chinese Hams Yuichi cell CHO
  • CH O dh fr gene-deficient Chinese hamster cell
  • Mouse L cells mouse AtT—20, mouse myeloid cells, rat GH3, mouse fibroblasts 3T3—L1, human liver cancer cells HepG2 (hereinafter referred to as HepG2 cells).
  • MG-63 human osteosarcoma cells
  • FL cells white adipocytes
  • egg cells ES cells
  • ES cells Evans, MJ and Kaufman, ⁇ . (1981) Nature, 292, 154.
  • mammalian stem cells or ES cells derived from mammals and particularly preferred are mouse ES cells, rat ES cells and human ES cells.
  • Methods for transforming these cells include the calcium phosphate method (Graham et al. (1973) Virology, 52,456 Elect-mouth poration method (Ishizaki et al. (1986) Cell Engineering, 5, 577), microinjection method, lipid for gene transfer. (Lipofectamine, Lipofectin; Gibco-BRL), etc. More specifically, To transform animal cells, see, for example, Cell Engineering Separate Volume 8 New Cell Engineering Experiment Protocol. 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973). It can be performed according to the method described.
  • the transformant is cultured in the presence of a specific compound, and the ability to control the promoter activity of the compound can be known by measuring and comparing the amount of the gene product in the culture.
  • the transformant is cultured by a method known per se.
  • the pH of the medium is preferably adjusted to about 5-8.
  • the recombinant vector of the present invention is a DNA containing the ECAT4 enhancer or the promoter region of ECAT4, the above transformant can be used to obtain the ECAT4 enhancer or ECAT4 promoter. Compounds that regulate (activate / suppress) the activity of the region can be screened.
  • the recombinant vector of the present invention is a DNA containing a consensus sequence recognized by ECAT4, the use of the above transformant controls the binding of ECAT4 to the consensus sequence (activation Z suppression). ) Can be used for screening compounds.
  • a substance that activates or suppresses ECAT4 expression can be obtained by introducing a chimeric gene in which a luciferase gene is linked to the base sequence of the ECAT4 enhancer / promoter region into cells (for example, ES cells). You can screen. Similarly, by introducing a chimeric gene of a promoter gene containing a consensus sequence recognized by ECAT4 and a repo overnight gene into cells (for example, ES cells, etc.), transcriptional activation or transcription by ECAT4 is performed. Substances that affect suppression or have the same activity as ECAT4 can be screened.
  • a well-known Binding Attach for example, SPA method (Amersham Armasia) It is also possible to search for a substance that inhibits the binding between ECAT4 and the consensus sequence of the present invention using the binding assay or the like used.
  • the method of screening for a substance that controls the activity of the ECAT4 enhancer or ECAT4 promoter region of the present invention includes the following steps (a), (b) and (c):
  • the transformant of the present invention described in the above (3) is useful.
  • test substance (candidate substance) to be screened by the screening method of the present invention is not limited, but may be a nucleic acid (including the antisense nucleotide of the present invention), a peptide, a protein, an organic compound, an inorganic compound, or the like.
  • the screening of the present invention is specifically carried out by bringing these test substances or a sample containing them (test sample) into contact with the cells.
  • test samples include, but are not limited to, a cell extract containing a test substance, an expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, a natural compound, and the like.
  • the conditions under which the test substance is brought into contact with the cells are not particularly limited, but it is preferable to select culture conditions (temperature, pH, medium composition, etc.) that do not kill the cells.
  • the screening method of the present invention includes a method of searching for a substance that controls the activity (suppression of activation Z) using the transcription ability of the ECAT4 enhancer promoter region as an index.
  • candidate substances or ES cells that are effective for maintaining ES cells in culture can be identified.
  • candidate substances effective for differentiation can be provided. It is considered that a candidate substance that activates the transcription ability of the ECAT4 enhancer region is useful for maintaining ES cells in culture, and a suppressor substance is useful for differentiating ES cells.
  • the activity of the ECAT4 enhancer / promoter region in cells to which the test substance (candidate substance) is added fluctuates (increases) compared to the level of cells in which the test substance (candidate substance) is not added. / Lower) can be selected.
  • the activity of the EC AT4 enhancer / promoter region is determined by connecting a marker gene such as a luciferase gene to a gene region (expression control region) that regulates the expression of the ECAT4 gene. It can be carried out by measuring the activity of a protein derived from the marker gene using a cell line into which the fusion gene has been introduced. Examples of the measuring method include, for example, measuring luciferase activity by a method according to the description of Brasier, A.R., et al. (1989) Biotecniques vol.7, 1116-1122.
  • the marker gene is preferably a structural gene of an enzyme that catalyzes a luminescence reaction or a color reaction.
  • a secreted alkaline phosphatase gene in addition to the luciferase gene, a secreted alkaline phosphatase gene, a chloramphenicol-acetyltransferase gene, a ⁇ -dalc-mouth nidase gene, Reporter genes such as the galactosidase gene and the aequorin gene can be exemplified.
  • EST expression sequence tag
  • the libraries used as representatives of various somatic cell tissues are # 467, 318, 198, 408, 239, 400, 453, 109, 16, 523, 161, 483, 258, 264, 419, 529, 327, 41 1, 417, 418, 399, 196, 271, 255, 495, 101, 98, 351, 416, 321, 251, 412, 379, 549, 329, 265, 449, 328, 516, 320, 436, 427 , 297, 366, 390, 315, 228, 277, 292, 284, 285 and 30 (total of 1328835 entries).
  • ES cell-specific marker genes such as Oct 3/4, UTF 1 (Okud aeta 1., 1998), and Re 1 (Roerseta 1., 1991). Exist, demonstrating the usefulness of this technology.
  • Northern blot analysis confirmed ES cell-specific expression of at least nine genes whose characteristics were not known.Here, one of them, which has a homeobox domain, is called ECAT4. (Fig. 1 (A)).
  • the 2184 base full-length cDNA of ECAT4 was isolated by the 5 'RACE method (Fig. 1 (A)). And a single gene encoding a 305 amino acid polypeptide.
  • valine is present at a position corresponding to tyrosine, which is strictly conserved in the NK-2 family.
  • the TN-2 and NK-2 specific domains that are conserved in the NK-2 family are absent from ECAT4. From these data EC
  • AT4 is not a NK-2 family.
  • ECAT4 is Oct 3
  • ECAT4 is considered to be a new homebox transcription factor that does not belong to any of the known subfamilies.
  • Exon 2 of the mouse ECAT4 gene having a homeobox domain was replaced with IRES (internal ribosome binding site) - ⁇ -geo (fusion of j3 galactosidase and neomycin resistance gene) Cassette (Mound tfordeta 1., 19994)
  • IRES internal ribosome binding site
  • - ⁇ -geo fusion of j3 galactosidase and neomycin resistance gene
  • Cassette (Mound tfordeta 1., 19994)
  • a targeting vector for replacing the hygromycin resistance gene was prepared.
  • a 4 kb fragment containing intron 1 and a 1.5 kbp fragment from exon 3 to exon 4 were amplified from the BAC clone and used as the 5 'and 3' homology regions of the targeting vector (Fig. 2 (A )).
  • the obtained evening targeting vector was introduced into RF8 ES cells by electroporation (Meinereta
  • the hygr vector was introduced into heterozygous mutant cells established using the / 3-geo vector.
  • Cells were sorted on MEF cells using LIF using hygromycin.
  • homologous recombination of the hygr vector was confirmed.
  • 11 clones were found to be homozygous for the ECAT4 mutation, both by Southern blot (FIG. 2 (B)) and PCR (FIG. 2 (C)).
  • Northern plot ( Figure 2 (D)) analysis confirmed the lack of ECAT4 expression in homozygous cells.
  • the hygr vector replaced the] 3-geo cassette.
  • the iS-geo vector was introduced into the heterozygous mutant cells established by the hygromycin vector.
  • Cells were sorted using G418 on SIF feeders expressing LIF.
  • 114 homologous recombination of the i3-geo vector was confirmed.
  • six clones are homozygous. It turned out. ECAT4-deficient cells obtained by both combinations exhibited the behavior as shown in Examples 4 and 5 below.
  • Hybridization using a 470 bp probe from the 5 'flanking region resulted in an 8.8 kb band from the wild-type locus, 11.4 kb from the target locus using the ⁇ geo target vector.
  • a 9.8 kb band was generated from the target locus using the hygromycin vector. Digestion was performed using Sca1 for 3 'Southern plot analysis.
  • Hybridization using a 1 kb probe from the 3 'flanking region resulted in an 11 kb band from the wild-type locus, and 1) a 15.4-kb band from the target locus using the 3 geo target vector.
  • a 7.3 kb band was generated from the target locus using the hygromycin vector.
  • the gene type of the mouse was determined using the three primers PCR.
  • the first sense primer, 6047-S5 was designed based on exon 2 to amplify the wild-type loci.
  • the second sense primer] 3—geoscreening 1 was designed based on the j8 geo cassette to amplify the targeted locus.
  • the common antisense primer 6047-int AS3 was designed based on intron 2 to amplify both wild-type and targeted loci. PCR using these primers resulted in an 850 bp fragment from the wild-type locus. A 600 bp fragment was generated from the targeted locus.
  • PCR was performed using ExTaq (Takara) according to the manufacturer's protocol.
  • the PCR program included initial denaturation (94 ° (: 2 minutes), 35 cycles (94 ° C 30 seconds, 53 ° C 30 seconds, 68X: 1 minute) and final extension (68 ° C 7 minutes).
  • the PCR program included initial denaturation (94 °
  • ECAT4 cells showed GATA4, GATA6, H inf 1/3, H inf 4 «, Coup—tfl, Coup- Primitive endoderm transcription factors such as tf II, laminin B1, dis ablled homolog 2 (Dab 2), Sp arc, tissue plasminogen activator (tPA), thrompomodulin (TM), etc.
  • tf II laminin B1, dis ablled homolog 2
  • Dab 2 dis ablled homolog 2
  • tPA tissue plasminogen activator
  • TM thrompomodulin
  • ECAT4 cells showed GATA4, GATA6, H inf 1/3, H inf 4 «, Coup—tfl, Coup- Primitive endoderm transcription factors such as tf II, laminin B1, dis ablled homolog 2 (Dab 2), Sp arc, tissue plasminogen activator (tPA), thrompomodulin (TM), etc.
  • AFP ⁇ -fetoprotein
  • BMP 2 bone
  • the mesodermal marker T. trophoblastoma — force Cdx 2, the primitive ectodermal force fibroblast growth factor (Fg f) — 5, or the neuroectodermal marker islet — 1 (I s 1 1) did not increase.
  • ECAT4-deficient cells had lost pluripotency
  • these cells were injected into blastocysts of C57BL / 6 mice.
  • the heterozygous mutant cells were injected, a plurality of chimeric mice to which ES cells greatly contributed, as judged from the color of the fur, were obtained.
  • chimera mice were not obtained when ECAT4 homozygous mutant cells were injected, confirming that the pluripotency of these cells was lost.
  • ECAT4 heterozygous ES cells obtained using the j3-geo vector were injected into C57BLZ6 blastocysts. Germline transmission was obtained from three independent ES clones. ECAT4 heterozygous mice were obtained at the expected Mendelian ratio, overall appearance was normal and reproductive. When the embryo before implantation was stained with X-ga1, expression of; 8-geo was detected only in the inner cell mass of the blastocyst (FIG. 4 (A)). Heterozygous crosses did not produce any homozygous offspring, indicating embryonic lethality.
  • the coding region of ECAT4 was introduced into the pCAG-IP vector to construct a gene vector.
  • the transgene vector was introduced into MG1 • 19 ES cells using Lip efo ctami n 2000 (Invitrogen) according to the protocol for ES cells provided by the manufacturer. Specifically, MG1.19 cells were seeded in a 6-well plate (1.2 x 10 5 cells swell), cultured for 24 hours, and 8 g of the transgene was transformed with 41 Lipofect ami n 2000. After forming the complex, it was added to the medium. The next day, the cells were seeded on a 100 mm Petri dish and selected using 2/2 g / ml puromycin. Sorting continued throughout the experiment.
  • the target gene is located downstream of a single CAG promoter. And the bite-mycin resistance gene are continuously present, and mRNA containing the two genes is transcribed. There is an internal ribosome entry site between the two, from which the puromycin resistance gene is translated. Therefore, all the cells that have become puromycin resistant will also express the target gene.
  • ECAT4 was introduced into MG1.19 cells, and puromycin was selected. As a result, 90% or more of the cells showed ECAT4 expression.
  • the resulting CAG-EC AT4 cells express significantly more EC AT4 transcripts (Fig. 5 (A)) and proteins (Fig. 5 (B)) compared to control cells transfected with the parental vector. did.
  • the pCAG-IP vector is an expression vector that is maintained extrachromosomally, and is known to disappear when culture is continued in the absence of Pyuguchimycin.
  • CAG-ECAT4 cells were cultured for 1 month in the absence of pure mouth mycin and in the presence of LIF. The disappearance of foreign gene expression was confirmed by Western plot. These cells differentiated like normal cells after removal of LIF. When CAG-ECAT4 cells were microinjected into blast cells, chimeric mice were formed. Therefore, it was confirmed that even if the cells overexpressing ECAT4 were cultured in the absence of LIF, removal of ECAT4 could regain the totipotency. Since the undifferentiated state of ES cells is sufficiently maintained even in the absence of LIF, the CAG-ECAT4 cells can be used to produce genetically modified animals. Break Variations are easier to make.
  • a fusion protein consisting of a maltose binding protein and ECAT4.
  • the coding region of the mouse ECAT gene was introduced into pIH1119 (provided from New England and Bio1 abs), and a transgene vector (pIH 11 19—ECAT4) was constructed.
  • This plasmid pIH1119-ECAT4 was introduced into E.coli strain BL21AI (Invitrogen) to obtain a transformant expressing MBP-ECAT4.
  • the cell extract was made up of a protease inhibitor cocktail (Nacalai Tesque) and 5 ml of a lysis buffer (2 OmM Tris-HCl (pH 7.4) containing 0.5 mg / ml of 1 ys0zyme, 20 OmM NaCl, ImM EDTA).
  • SELEX was implemented as follows. For the synthesis of double-stranded oligonucleotides, lg SELEX—N2O-01 igo (SEQ ID NO: 11), 1 xg SELEX—N20 RV primer (SEQ ID NO: 12), 2.5 1 10 x Kle no w The reaction was performed in a reaction solution containing buffer, 41 10 mM dNTPs, and 14.7 ⁇ 1 purified water. Denaturation of the mixture was performed at 95 ° C for 5 minutes, and annealing was performed at 54 ° C for 5 minutes.
  • a K1 enow fragment (3.611 x 0.225 U in K1enow buffer) was added to the mixture, which was then incubated at 25 ° C for 40 minutes.
  • the double-stranded DNA was purified by phenol Z-cloth form extraction and ethanol precipitation and resuspended in 20 n1 purified water.
  • Performing DNA-protein binding requires 201 D NA, 301 MB P—EC AT 4—binding beads, 201 s 5 ⁇ binding buffer (l O OmM HEPES—K ⁇ H, pH 7.9, lmM. EDTA, 1M KC 1, and 50% (Glycerol) and 301 water at 4 ° C. for 30 minutes. Washing of the beads was performed 6 times using 100 1 lysis buffer supplemented with ImM 0 and 0.2 mM PMSF.
  • DNA was purified by phenol / mouth opening form extraction and ethanol precipitation and resuspended in 201 purified water. PCR amplification was performed using this 51, and the reaction solution at this time was 51 1 OxExTaq buffer, 4/1 1 2.5 mM dNT P, 1 1 30 1 ⁇ SELEX— It contained N20 FW primer (SEQ ID NO: 13), 1 a1 of 30 MS ELEX-N20 RV primer, 0 of ExTad polymerase and 33.75 L of water. The amplification product was purified using a Micro-bio spin column (BioRad), concentrated to 201 by ethanol precipitation, and half of it was advanced to the next round. This procedure was repeated five times for concentration.
  • BioRad Micro-bio spin column
  • the amplification product in the final eluate of the oligonucleotide that specifically binds to the MBP-ECAT4 fusion protein thus obtained was ligated into a PCR2.1 vector (Invitrogen). Forty clones were randomly selected and sequenced.
  • MBP maltose binding protein
  • E.coli maltose binding protein
  • a 20-mer random oligonucleotide with an adapter sequence at each end was applied to the MBP-ECAT4 binding resin and washed thoroughly.
  • the DNA bound to the resin was amplified by PCR using a primer corresponding to the adapter sequence.
  • the amplification product was applied to a new MBP-ECAT4 column. This procedure was repeated five times to enrich for oligonucleotides that specifically bind to the MBP-ECAT4 fusion protein.
  • the amplification product obtained from the final reaction was subcloned into the pCR2.1 vector for sequence analysis.
  • an ECAT4 consensus sequence was identified approximately 4 kb upstream of the transcription start site in the 5'-flanking region of the mouse GATA6 gene (Fig. 6 (B), sequence number 5). This region is known to be essential for the expression of GAT A6 in the heart (Molkintineta 1., 2000; Sun-Wadaeta 1., 2000). Comparing the genomic sequences of the mouse and human GATA6 genes, this region was found to be highly conserved. The human sequence also contained the ECAT4 consensus ( Figure 6 (B), SEQ ID NO: 6).
  • an ES cell in which ECAT4 is forcibly expressed is provided. Furthermore, the present invention also provides a consensus sequence for ECAT4, an ECAT4 enhancer, and uses thereof. Since the function of ECAT4 as a transcription factor promoting self-renewal of ES cells has been clearly demonstrated, they are extremely useful in the research and development of ES cells. Sequence listing free text
  • the base sequence described in SEQ ID NO: 4 is a consensus sequence recognized by ECAT4.
  • the base sequence described in SEQ ID NO: 11 is SELEX-N20-Oiigo.
  • the base sequence described in SEQ ID NO: 12 is a SE LEX-N20RV primer.
  • the base sequence described in SEQ ID NO: 13 is a SELEX-N20 FW primer.

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Abstract

L'objet de la présente invention se rapporte à la production d'une cellule SE dont l'expression de ECAT4 est forcée. La cellule SE présentant une expression forcée de ECAT4 peut être cultivée en l'absence de cellules nourricières ou LIF. On cherche également à produire une séquence consensus reconnue par ECAT4, un activateur ECAT4, une région promoteur ECAT4 ainsi que son utilisation. Etant donné qu'il est clairement indiqué que ECAT4 possède une fonctionnalité en tant que facteur de transcription stimulant l'autoréplication de cellules SE, on lui trouve une grande utilité dans le domaine de la recherche et de la mise au point de cellules SE.
PCT/JP2004/000790 2003-01-31 2004-01-28 Facteur de determination de l'autoreplication de cellules souches embryonnaires WO2004067744A1 (fr)

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Cited By (3)

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WO2006035741A1 (fr) * 2004-09-29 2006-04-06 Dainippon Sumitomo Pharma Co., Ltd. Gène exprimé spécifiquement dans les cellules es et utilisation dudit gène
EP1802744A1 (fr) * 2004-09-03 2007-07-04 Agency for Science, Technology and Research Procédé de maintien de pluripotence de cellules souches/progénitrices
JP2010522565A (ja) * 2007-03-26 2010-07-08 アメリカ合衆国 胚性幹細胞分化を調整する方法

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WO2003064463A2 (fr) * 2002-01-30 2003-08-07 The University Of Edinburgh Facteurs determinant la pluripotence et utilisations de ces facteurs

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WO2003064463A2 (fr) * 2002-01-30 2003-08-07 The University Of Edinburgh Facteurs determinant la pluripotence et utilisations de ces facteurs

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1802744A1 (fr) * 2004-09-03 2007-07-04 Agency for Science, Technology and Research Procédé de maintien de pluripotence de cellules souches/progénitrices
EP1802744A4 (fr) * 2004-09-03 2008-06-18 Agency Science Tech & Res Procédé de maintien de pluripotence de cellules souches/progénitrices
WO2006035741A1 (fr) * 2004-09-29 2006-04-06 Dainippon Sumitomo Pharma Co., Ltd. Gène exprimé spécifiquement dans les cellules es et utilisation dudit gène
JPWO2006035741A1 (ja) * 2004-09-29 2008-05-15 伸弥 山中 Ｅｓ細胞特異的発現遺伝子及びその利用
US7803920B2 (en) 2004-09-29 2010-09-28 Shinya Yamanaka ECAT16 gene expressed specifically in ES cells and utilization of the same
JP2010522565A (ja) * 2007-03-26 2010-07-08 アメリカ合衆国 胚性幹細胞分化を調整する方法
US8617813B2 (en) 2007-03-26 2013-12-31 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Methods for modulating embryonic stem cell differentiation

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