Mutant loxP site and applications thereof

• US 6,465,254 B1
• Filed: 07/07/2000
• Issued: 10/15/2002
• Est. Priority Date: 11/13/1997
• Status: Expired due to Term

• Current Assignee: Dainippon Sumitomo Pharma Company Limited (Sumitomo Chemical Company Limited)
• Original Assignee: Sumitomo Pharmaceuticals Company Limited (Sumitomo Chemical Company Limited)
• Inventors: Tanaka, Keiji, Saito, Izumu
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Li, Janice
• Application Number:
  US09/554,271
• Time in Patent Office: 830 Days
• Field of Search: 435/455, 435/462, 435/463, 800/21, 424/93.21, 536/231
• US Class Current: 435/462
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2217/075 inducing loss of function, i.e. knock out
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Abstract

Highly efficient gene integration or gene replacement in the higher eucaryote including animal cells can be performed by using mutant loxP site having the following properties (a)-(c) in the present invention.

(a) a nucleotide sequence wherein, in a wild-type loxP site of the following formula (SEQ ID NO: 1) derived from E. coli P1 phage, at least one of the bases consisting of second (T), third (G), fourth (T) and fifth (A) bases, and at least one of the bases consisting of sixth (T) and seventh (G) bases within the 8 bases in the central part of the sequence (spacer region) are substituted by different base, and regions except for the spacer region are optionally substituted by any base:

                 12345678 5′-ATAACTTCGTATA ATGTATGC TATACGAAGTTAT-3′                Spacer Region

(b) a specific recombination between said mutant loxP and the wild-type loxP site can not occur even in the presence of recombinase Cre; and

(c) a specific recombination between the mutant loxP sites having identical nucleotide sequences can occur in the presence of recombinase Cre.

Claims

1. A mutant loxP site of a wild-type loxP site derived from E. coli P1 phage represented by SEQ ID NO:

1

wherein in said mutant loxP site, one nucleotide base in SEQ ID NO;

1 selected from the group consisting of

T at position 2 in the spacer,

G at position 3 in the spacer,

T at position 4 in the spacer, and

A at position 5 in the spacer,

is substituted by a different nucleotide base; and

wherein in said mutant loxP site, one nucleotide base in SEQ ID NO;

1 selected from the group consisting of

T at position 6 in the spacer, and

G at position 7 in the spacer,

is substituted by a different nucleotide base;

wherein, even in the presence of recombinase Cre, said mutant loxP site does not recombine with a wild-type loxP site derived from E. coli P1 phage via a specific DNA recombination event between said mutant loxP site and a wild-type loxP site derived from E. coli P1 phage; and

wherein, in the presence of recombinase Cre, said mutant loxP site recombines with a second mutant loxP site which has an identical sequence as said mutant loxP site via a specific DNA recombination event between said mutant loxP site and said second mutant loxP site.

2. The mutant loxP site of claim 1, wherein one or both of said inverted repeats contains at least one nucleotide base substitution.

5. The mutant loxP site of claim 1, wherein, in the presence of recombinase Cre, said mutant loxP site does not recombine with a second mutant loxP site which has a different sequence than said mutant loxP site, via a specific DNA recombination event between said mutant loxP site and said second mutant loxP site.

7. The mutant loxP site of claim 1, wherein said mutant loxP site is represented by a member selected from the group consisting of SEQ ID NO:

26, SEQ ID NO;

29, SEQ ID NO;

30, SEQ ID NO;

35, SEQ ID NO;

39, SEQ ID NO;

42, and SEQ ID NO;

49.

8. An isolated DNA molecule comprising the mutant loxP site of any one of claims 1 to 7.

9. An isolated DNA molecule comprising at least one wild-type loxP site derived from E. coli P1 phage and at least one mutant loxP site of claim 1.

11. The isolated DNA molecule of claim 9, further comprising a desired DNA molecule inserted between the wild-type loxP site derived from E. coli P1 phage and the mutant loxP site.

13. An isolated DNA molecule comprising at least two mutant loxP sites of claim 5, wherein each mutant loxP site has a different nucleotide sequence.

15. The isolated DNA molecule of claim 13, further comprising a desired DNA molecule inserted between the two different mutant loxP sites.

18. A method for replacing a desired DNA molecule comprising contacting, in the presence of recombinase Cre,

DNA molecule (A) comprising a first desired DNA molecule inserted between a wild-type loxP site derived from E. coli P1 phage and a mutant loxP site of claim 1 or 3, with

circular DNA molecule (B) comprising a second desired DNA molecule inserted between a wild-type loxP site derived from E. coli P1 phage and a mutant loxP site having the same DNA sequence as the mutant loxP site of said DNA molecule (A),

so as to cause a recombinational event whereby said first desired DNA molecule in said DNA molecule (A) is replaced by said second desired DNA molecule,

wherein said first desired DNA molecule is different from said second desired DNA molecule.

19. A method for replacing a desired DNA molecule comprising contacting, in the presence of recombinase Cre,

DNA molecule (A) comprising a first desired DNA molecule inserted between a first mutant loxP site of claim 5 or 6 and a second mutant loxP site of claim 5 or 6, wherein the sequence of said first mutant loxP site is different from the sequence of said second mutant loxP site, with

circular DNA molecule (B) comprising a second desired DNA molecule inserted between a mutant loxP site having the same DNA sequence as the first mutant loxP site of said DNA molecule (A) and a second mutant loxP site having the same DNA sequences as the second mutant loxP site of said DNA molecule (A),

so as to cause a recombinational event whereby said first desired DNA molecule in said DNA molecule (A) is replaced by said second desired DNA molecule,

wherein said first desired DNA molecule is different from said second desired DNA molecule.

20. The method of claim 18, wherein said second desired DNA molecule is not a functional gene.

21. The method of claim 19, wherein said second desired DNA molecule is not a functional gene.

22. The method of claim 18, wherein said first desired DNA molecule is not a functional gene.

23. The method of claim 19, wherein said first desired DNA molecule is not a functional gene.

24. The method of claim 18, wherein said DNA molecule (A) is chromosomal DNA of a cell, and said circular DNA molecule (B) is plasmid DNA or DNA of a double-stranded circular DNA virus.

25. The method of claim 19, wherein said DNA molecule (A) is chromosomal DNA of a cell, and said circular DNA molecule (B) is plasmid DNA or DNA of a double-stranded circular DNA virus.

26. The method of claim 18, wherein said DNA molecule (A) is chromosomal DNA of a cell, and said circular DNA molecule (B) is converted, intracellularly, to double-stranded circular DNA.

27. The method of claim 19, wherein said DNA molecule (A) is chromosomal DNA of a cell, and said circular DNA molecule (B) is converted, intracellularly, to double-stranded circular DNA.

28. The method of claim 18, wherein said DNA molecule (A) is genomic DNA of a double-stranded DNA virus, and said circular DNA molecule (B) is plasmid DNA or DNA of a double-stranded circular DNA virus.

29. The method of claim 19, wherein said DNA molecule (A) is genomic DNA of a double-stranded DNA virus, and said circular DNA molecule (B) is plasmid DNA or DNA of a double-stranded circular DNA virus.

30. The method of claim 18, wherein said DNA molecule (A) is genomic DNA of a double-stranded DNA virus, and said circular DNA molecule (B) is converted, intracellularly, to double-stranded circular DNA.

31. The method of claim 19, wherein said DNA molecule (A) is genomic DNA of a double-stranded DNA virus, and said circular DNA molecule (B) is converted, intracellularly, to double-stranded circular DNA.

32. The method of claim 28, wherein said double-stranded DNA virus is adenovirus.

33. The method of claim 29, wherein said double-stranded DNA virus is adenovirus.

3. A mutant loxP site of a wild-type loxP site derived from E. coli P1 phage represented by SEQ ID NO:

1

wherein in said mutant loxP site, one nucleotide base in SEQ ID NO;

1 selected from the group consisting of

T at position 2 in the spacer,

G at position 3 in the spacer, and

T at position 4 in the spacer,

is substituted by a different nucleotide base;

wherein, even in the presence of recombinase Cre, said mutant loxP site does not recombine with a wild-type loxP site derived from E. coli P1 phage via a specific DNA recombination event between said mutant loxP site and a wild-type loxP site derived from E. coli P1 phage; and

wherein, in the presence of recombinase Cre, said mutant loxP site recombines with a second mutant loxP site which has an identical sequence as said mutant loxP site via a specific DNA recombination event between said mutant loxP site and said second mutant loxP site.

4. The mutant loxP site of claim 3, wherein one or both of said inverted repeats contains at least one nucleotide base substitution.

6. The mutant loxP site of claim 3, wherein, in the presence of recombinase Cre, said mutant loxP site does not recombine with a second mutant loxP site which has a different sequence than said mutant loxP site, via a specific DNA recombination event between said mutant loxP site and said second mutant loxP site.

10. An isolated DNA molecule comprising at least one wild-type loxP site derived from E. coli P1 phage and at least one mutant loxP site of claim 3.

12. The isolated DNA molecule of claim 10, further comprising a desired DNA molecule inserted between the wild-type loxP site derived from E. coli P1 phage and the mutant loxP site.

14. An isolated DNA molecule comprising at least two mutant loxP sites of claim 6, wherein each mutant loxP site has a different nucleotide sequence.

16. The isolated DNA molecule of claim 14, further comprising a desired DNA molecule inserted between the two different mutant loxP sites.

17. A cell which is transformed with the isolated DNA molecule of any one of claims 9 to 16.

34. A composition comprising the DNA molecule of any one of claims 9 to 16, and a pharmaceutically acceptable carrier or diluent.