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Method for transcription/degradation dual control of protein by antibiotic

外国特許コード F110005398
整理番号 K02213WO
掲載日 2011年9月5日
出願国 アメリカ合衆国
出願番号 53111008
公報番号 20100136654
公報番号 8232099
出願日 平成20年3月14日(2008.3.14)
公報発行日 平成22年6月3日(2010.6.3)
公報発行日 平成24年7月31日(2012.7.31)
国際出願番号 JP2008055200
国際公開番号 WO2008114856
国際出願日 平成20年3月14日(2008.3.14)
国際公開日 平成20年9月25日(2008.9.25)
優先権データ
  • 特願2007-065415 (2007.3.14) JP
  • 2008WO-JP55200 (2008.3.14) WO
発明の名称 (英語) Method for transcription/degradation dual control of protein by antibiotic
発明の概要(英語) (US8232099)
The present invention provides an expression vector, containing expressibly (a) a polynucleotide encoding a fusion protein of a mutant of a repressor protein, which binds to an antibiotic, and a target protein, and (b) a polynucleotide encoding a protein controlling the transcription of the polynucleotide in (a), the transcription of the polynucleotide in (a) and the degradation of said fusion protein, which is the expression product of the polynucleotide in (a), being controlled inside a cell by the presence or absence of an antibiotic inside the cell.
特許請求の範囲(英語) [claim1]
1. A gene expression control system for controlling the expression of a target gene inside a cell in vitro or in a non-human transgenic animal by the presence or absence of a tetracycline antibiotic, comprising: (a) a cell
(b) an expression vector that is introduced into the interior of said cell, and comprises expressibly (b1) a polynucleotide encoding a fusion protein of a mutant of a tetracycline repressor protein and a recombination enzyme, wherein said fusion protein mediates the recombination at a recombination sequence site, and is degraded inside said cell in the absence of said antibiotic,
wherein said mutant has an amino acid sequence having at least two of the following mutations: substitution of aspartic acid at position 95 with asparagine, substitution of leucine at position 101 with serine and substitution of glycine at 102 with aspartic acid in the amino acid sequence of a wild-type tetracycline repressor protein encoded by SEQ ID NO: 2, and
(b2) a polynucleotide to be introduced into the interior of said cell, encoding a protein that binds to the transcription control region of the polynucleotide in (b1) and controls the transcription of said polynucleotide, wherein the binding to said transcription control region is controlled by the presence or absence of said antibiotic,
(c) an expression vector that is introduced into the interior of said cell, and comprises expressibly a target gene between and/or downstream of recombination sequences, and
(d) a tetracycline antibiotic to be introduced into the interior of said cell, wherein the transcription of the polynucleotide in (b1) and the degradation of said fusion protein, which is the expression product of the polynucleotide in (b1), is controlled inside the cell by the presence or absence of the tetracycline antibiotic, and the expression of said target gene is controlled by the expressed amount of said fusion protein.
[claim2]
2. The system according to claim 1, wherein said recombination enzyme is at least one protein selected from the group consisting of: (a) Cre recombinase;
(b) FLP recombinase;
(c) phage phi 13 integrase;
(d) phage R4 integrase;
(e) phage TP901-1 integrase;
(f) phage lambda (lambda) integrase;
(g) phage HK022 integrase;
(h) beta (beta) recombinase;
(i) R recombinase;
(j) gamma delta (gamma delta) resolvase;
(k) Dre recombinase;
(l) phi Rv1 integrase;
(m) Int;
(n) IHF;
(o) Xis;
(p) Fis;
(q) Hin;
(r) Gin;
(s) Cin;
(t) Th3 resolvase;
(u) TndX;
(v) XerC; and
(w) XerD.
[claim3]
3. The system according to claim 1, wherein said recombination sequence comprises one or more recombination sequences selected from the group consisting of: (a) loxP;
(b) frt;
(c) attB/attP;
(d) six;
(e) RS;
(f) res;
(g) rox;
(h) psi;
(i) dif;
(j) cer; and
(k) mutants, variants, and derivatives of the recombination sequence from (a), (b), (c), (d), (e), (f), (g), (h), (i) or (j), which have retained the capability of provoking recombination.
[claim4]
4. The system according to claim 1, wherein said recombination enzyme is Cre recombinase and said recombination sequence is the loxP sequence.
[claim5]
5. The system according to claim 1, wherein said target gene is a transcription factor.
[claim6]
6. A gene expression control method for controlling the expression of a target gene inside a cell in vitro or in a non-human transgenic animal by the presence or absence of a tetracycline antibiotic, comprising the step of expressing under the presence or under the absence of the antibiotic inside said cell, (a) an expression vector comprising expressibly (a1) a polynucleotide encoding a fusion protein of a mutant of a tetracycline repressor protein and a recombination enzyme, wherein said fusion protein mediates the recombination at a recombination sequence site, and is degraded inside said cell in the absence of said antibiotic,
wherein said mutant has an amino acid sequence having at least two of the following mutations: substitution of aspartic acid at position 95 with asparagine, substitution of leucine at position 101 with serine and substitution of glycine at 102 with aspartic acid in the amino acid sequence of a wild-type tetracycline repressor protein encoded by SEQ ID NO: 2, and
(a2) a polynucleotide encoding a protein that binds to the transcription control region of the polynucleotide in (a1) and controls the transcription of said polynucleotide, wherein the binding to said transcription control region is controlled by the presence or absence of said antibiotic, and
(b) an expression vector comprising expressibly a target gene between and/or downstream of recombination sequences.
[claim7]
7. The method according to claim 6, wherein said recombination enzyme is at least one protein selected from the group consisting of: (a) Cre recombinase;
(b) FLP recombinase;
(c) phage phi 13 integrase;
(d) phage R4 integrase;
(e) phage TP901-1 integrase;
(f) phage lambda (lambda) integrase;
(g) phage HK022 integrase;
(h) beta (beta) recombinase;
(i) R recombinase;
(i) gamma delta (gamma delta) resolvase;
(k) Dre recombinase;
(l) phi Rv1 integrase
(m) Int;
(n) IHF;
(o) Xis;
(p) Fis;
(q) Hin;
(r) Gin;
(s) Cin;
(t) Th3 resolvase;
(u) TndX;
(v) XerC; and
(w) XerD.
[claim8]
8. The method according to claim 6, wherein said recombination sequence comprises one or more recombination sequences selected from the group consisting of: (a) loxP;
(b) frt;
(c) attB/attP;
(d) six;
(e) RS;
(f) res;
(g) rox;
(h) psi;
(i) dif;
(j) cer; and
(k) mutants, variants, and derivatives of the recombination sequence from (a), (b), (c), (d), (e), (f), (g), (h), (i) or (j), which have retained the capability of provoking recombination.
[claim9]
9. The method according to claim 6, wherein said recombination enzyme is Cre recombinase and the recombination sequence is the loxP sequence.
[claim10]
10. The method according to claim 6, wherein said target gene is a transcription factor.
[claim11]
11. The system according to claim 1, wherein said expression vector in (b) comprises a polynucleotide encoding a polypeptide comprising a nuclear export sequence added to the C-terminal side of the amino acid sequence of the fusion protein in (b1).
[claim12]
12. The system according to claim 5, wherein said transcription factor is any of Oct3/4, Klf4, Sox2, or c-Myc gene.
[claim13]
13. The method according to claim 6, wherein said expression vector in (a) comprises a polynucleotide encoding a polypeptide comprising a nuclear export sequence added to the C-terminal side of the amino acid sequence of the fusion protein in (a1).
[claim14]
14. A gene expression control system for controlling the expression of a target gene inside a cell in vitro or in a non-human transgenic animal by the presence or absence of a tetracycline antibiotic, comprising (a) cell
(b) an expression vector that is introduced into the interior of said cell, and comprises expressibly a polynucleotide encoding a fusion protein of a mutant of a tetracycline repressor protein and a recombination enzyme, wherein said fusion protein mediates the recombination at the recombination sequence site, and is degraded inside said cell in the absence of said antibiotic, wherein said mutant has an amino acid sequence having at least two of the following mutations: substitution of aspartic acid at position 95 with asparagine, substitution of leucine at position 101 with serine and substitution of glycine at 102 with aspartic acid in the amino acid sequence of a wild-type tetracycline repressor protein encoded by SEQ ID NO: 2,
(c) an expression vector that is introduced into the interior of said cell, and comprises expressibly a polynucleotide encoding a protein that binds to the transcription control region of the polynucleotide in (b) and controls the transcription of said polynucleotide, wherein the binding to said transcription control region is controlled by the presence or absence of said antibiotic,
(d) an expression vector that is introduced into the interior of said cell, and comprises expressibly a target gene between and/or downstream of recombination sequences, and
(e) said antibiotic to be introduced into the interior of said cell, wherein the transcription of the polynucleotide in (b) and the degradation of the fusion protein, which is the expression product of the polynucleotide in (b), is controlled inside said cell by the presence or absence of the antibiotic, and the expression of the target gene is controlled by the expressed amount of the fusion protein.
[claim15]
15. A gene expression control method for controlling the expression of a target gene inside a cell in vitro or in a non-human transgenic animal by the presence or absence of a tetracycline antibiotic, comprising the step of expressing, in the presence or under the absence of the antibiotic inside said cell, (a) an expression vector comprising expressibly a polynucleotide encoding a fusion protein of a mutant of a tetracycline repressor protein and a recombination enzyme, wherein said fusion protein mediates the recombination at the recombination sequence site, and is degraded inside said cell in the absence of said antibiotic, wherein said mutant has an amino acid sequence having at least two of the following mutations: substitution of aspartic acid at position 95 with asparagine, substitution of leucine at position 101 with serine and substitution of glycine at 102 with aspartic acid in the amino acid sequence of a wild-type tetracycline repressor protein encoded by SEQ ID NO: 2,
(b) an expression vector comprising expressibly a polynucleotide encoding a protein that binds to the transcription control region of the polynucleotide in (a) and controls the transcription of said polynucleotide, wherein the binding to said transcription control region is controlled by the presence or absence of said antibiotic, and
(c) an expression vector comprising expressibly a target gene between and/or downstream of recombination sequences.
  • 発明者/出願人(英語)
  • MIWA YOSHIHIRO
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
参考情報 (研究プロジェクト等) PRESTO Structure Function and Measurement Analysis AREA
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