Top > Search of International Patents > Method for of transcription-degradation dual regulation for protein by antibiotic

Method for of transcription-degradation dual regulation for protein by antibiotic

Foreign code F110004017
File No. K02213WO
Posted date Jul 8, 2011
Country EPO
Application number 08722565
Gazette No. 2135945
Gazette No. 2135945
Date of filing Mar 14, 2008
Gazette Date Dec 23, 2009
Gazette Date Apr 23, 2014
International application number JP2008055200
International publication number WO2008114856
Date of international filing Mar 14, 2008
Date of international publication Sep 25, 2008
Priority data
  • 2008JP055200 (Mar 14, 2008) WO
  • P2007-065415 (Mar 14, 2007) JP
Title Method for of transcription-degradation dual regulation for protein by antibiotic
Abstract 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.(see diagramm)
Scope of claims [claim1]
1. An expression vector for controlling the expression of a target gene inside a cell at the transcription level and the protein degradation level by the presence or absence of an antibiotic in the cell into which another expression vector containing expressibly the target gene between and/or downstream of recombination sequences has been introduced, comprising expressibly: (a) a polynucleotide encoding a fusion protein of a mutant of a repressor protein that binds to said antibiotic 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, and (b) 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.
[claim2]
2. The expression vector 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 expression vector according to Claim 1 or 2, wherein said recombination sequence contains 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 expression vector according to any of Claims 1 to 3, wherein said recombination enzyme is Cre recombinase and said recombination sequence is the loxP sequence.
[claim5]
5. The expression vector according to any of Claims 1 to 4, wherein said target gene is a transcription factor.
[claim6]
6. The expression vector according to Claim 5, wherein said transcription factor is any of Oct3/4, Klf4, Sox2 or c-Myc gene.
[claim7]
7. The expression vector according to any of Claims 1 to 6, wherein said polynucleotide in (b) encodes a protein that binds to the transcription control region of said polynucleotide in (a) and enhances the transcription of said polynucleotide, said protein being able to bind to said transcription control region only when bound to said antibiotic.
[claim8]
8. The expression vector according to any of Claims 1 to 7, wherein said antibiotic is a tetracycline-type antibiotic.
[claim9]
9. The expression vector according to Claim 8, wherein said tetracycline-type antibiotic is tetracycline, or, doxycycline, oxytetracycline, chlortetracycline or anhydrotetracycline, which are derivatives thereof.
[claim10]
10. The expression vector according to any of Claims 1 to 9, wherein said mutant of the repressor protein is a mutant of a tetracycline repressor protein.
[claim11]
11. The expression vector according to Claim 10, wherein said mutant of the tetracycline repressor protein has an amino acid sequence comprising at least one amino acid residue substituted in the amino acid sequence of a wild-type tetracycline repressor protein.
[claim12]
12. The expression vector according to Claim 11, wherein said substitution of amino acid residue is present at at least any two sites among the aspartic acid at position 95, the leucine at position 101 and the glycine at position 102 of the amino acid sequence of the wild-type tetracycline repressor protein.
[claim13]
13. A gene expression control system for controlling the expression of a target gene inside a cell by the presence or absence of an antibiotic, comprising (a) a cell (b) an expression vector according to claim 1 that is to be introduced into the interior of said cell, (c) an expression vector that is to be introduced into the interior of said cell, and contains expressibly a target gene between and/or downstream of recombination sequences, and (d) an antibiotic that is to be introduced into the interior of said cell,
wherein the transcription of the polynucleotide encoding a fusion protein in (b) and the degradation of said fusion protein, is controlled inside said cell by the presence or absence of the antibiotic, and the expression of said target gene is controlled by the expressed amount of said fusion protein.
[claim14]
14. The system according to Claim 13, 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.
[claim15]
15. The system according to Claim 13 or 14, wherein said recombination sequence contains 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.
[claim16]
16. The system according to any of Claims 13 to 15, wherein said recombination enzyme is Cre recombinase and said recombination sequence is the loxP sequence.
[claim17]
17. The system according to any of Claims 13 to 16, wherein said target gene is a transcription factor.
[claim18]
18. An in vitro gene expression control method for controlling the expression of a target gene inside a cell by the presence or absence of an antibiotic, comprising the step of expressing under the presence or under the absence of an antibiotic inside said cell, (a) an expression vector according to claim 1
and (b) an expression vector containing expressibly a target gene between and/or downstream of recombination sequences.
[claim19]
19. The method according to Claim 18, 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.
[claim20]
20. The method according to Claim 18 or 19, wherein said recombination sequence contains 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.
[claim21]
21. The method according to any of Claims 18 to 20, wherein said recombination enzyme is Cre recombinase and the recombination sequence is the loxP sequence.
[claim22]
22. The method according to any of Claims 18 to 21, wherein said target gene is a transcription factor.
[claim23]
23. The expression vector according to any of Claims 1 to 12, wherein said expression vector contains 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 (a).
[claim24]
24. A gene expression control kit for controlling the expression of a target gene inside a cell at the transcription level and the protein degradation level by the presence or absence of an antibiotic in the cell into which an expression vector containing expressibly the target gene between and/or downstream of recombination sequences has been introduced, said kit comprising: (a) an expression vector containing expressibly a polynucleotide encoding a fusion protein of a mutant of a repressor protein that binds to said antibiotic, 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, and (b) an expression vector containing 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.
[claim25]
25. A gene expression control system for controlling the expression of a target gene inside a cell by the presence or absence of an antibiotic, comprising (a) a cell (b) an expression vector according to claim 24(a) that is introduced into the interior of said cell, (c) an expression vector according to claim 24(b) that is introduced into the interior of said cell, (d) an expression vector that is introduced into the interior of said cell, and contains expressibly a target gene between and/or downstream of recombination sequences, and (e) said antibiotic to be introduced into the interior of said above 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.
[claim26]
26. An in vitro gene expression control method for controlling the expression of a target gene inside a cell by the presence or absence of an antibiotic, comprising the step of expressing, in the presence or under the absence of an antibiotic inside said cell, (a) an expression vector according to claim 24(a), (b) an expression vector according to claim 24(b), and (c) an expression vector containing expressibly a target gene between and/or downstream of recombination sequences.
  • Applicant
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • MIWA YOSHIHIRO
IPC(International Patent Classification)
Reference ( R and D project ) PRESTO Structure Function and Measurement Analysis AREA
Please contact us by E-mail or facsimile if you have any interests on this patent.

PAGE TOP

close
close
close
close
close
close