Site-specific recombination of DNA in eukaryotic cells

• US 4,959,317 A
• Filed: 04/29/1987
• Issued: 09/25/1990
• Est. Priority Date: 10/07/1985
• Status: Expired due to Term

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Sauer, Brian L.
• Primary Examiner(s): Teskin, Robin L.
• Application Number:
  US07/043,795
• Time in Patent Office: 1,245 Days
• Field of Search: 435/68, 435/70, 435/172.1, 435/172.3, 435/253, 435/317, 435/320, 435/256, 435/254, 435/255, 435/91, 435/240.2, 530/360, 536/27, 935/28, 935/34, 935/69, 935/37
• US Class Current: 435/462
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

A method for producing site-specific recombination of DNA in eukaryotic cells at regions designated lox sites is disclosed.

Claims

1. A method for producing site-specific recombination of DNA in eukaryotic cells, comprising,

(a) introducing into the cells a first DNA sequence comprising a first lox site and a second DNA sequence comprising a second lox site, and

(b) contacting the lox sites with Cre, thereby producing the site specific recombination.

2. A method as defined in claim 1, wherein a third DNA sequence comprising a cre gene is also introduced into the cells.

3. A method as defined in claim 2, wherein the third DNA sequence further comprises a regulatory nucleotide sequence and expression of the cre gene is produced by activating the regulatory nucleotide sequence.

4. A method as defined in claim 3, wherein the first and second DNA sequences are introduced into the cells connected by a pre-selected DNA segment.

5. A method as defined in claim 4, wherein the first and second lox sites have the same orientation and the site specific recombination of DNA is a deletion of the pre-selected DNA segment.

6. A method as defined in claim 5, wherein the cre gene is isolated from bacteriophage P1.

7. A method as defined in claim 5, wherein the first and second lox sites are loxP or loxC2.

8. A method as defined in claim 7, wherein the pre-selected DNA segment is a gene for a structural protein, an enzyme, or a regulatory molecule.

9. A method as defined in claim 8, wherein the eukaryotic cell is a mammalian cell.

10. A method as defined in claim 8, wherein the eukaryotic cell is yeast.

11. A method as defined in claim 4, wherein the first and second lox sites have opposite orientations and the site-specific recombination is an inversion of the nucleotide sequence of the pre-selected DNA segment.

12. A method as defined in claim 11, wherein the cre gene is isolated from bacteriophage P1.

13. A method as defined in claim 12, wherein the first and second lox sites are loxP or loxC2.

14. A method as defined in claim 13, wherein the pre-selected DNA segment is a gene for a structural protein, an enzyme, or a regulatory molecule.

15. A method as defined in claim 1, wherein the second and third DNA sequences are introduced into two different DNA molecules and the site-specific recombination is a reciprocal exchange of DNA segments proximate to the lox sites.

16. A method as defined in claim 15, wherein the cre gene is isolated from bacteriophage P1.

17. A method as defined in claim 16, wherein the first and second lox sites are loxP and loxC2.

21. A method as defined in claim 1, wherein the DNA sequences comprising lox sites are introduced into the DNA in yeast connected by a pre-selected DNA segment.

22. A method as defined in claim 21, wherein the cre gene is isolated from bacteriophage P1.

23. A method as defined in claim 22, wherein the fir t and second lox sites are loxP or loxC2.

18. A eukaryotic cell transformed with a DNA sequence comprising a cre gene.

19. A eukaryotic cell transformed with a DNA sequence comprising a lox site.

20. A method for producing site-specific recombination of DNA in yeast, comprising:

(a) introducing into the DNA the following DNA sequences;

(i) a DNA sequence comprising a regulatory nucleotide sequence and a cre gene,

(ii) a DNA sequence comprising a first lox site, and

(iii) a DNA sequence comprising a second lox site, and

(b) activating the regulatory nucleotide sequence thereby effecting expression of the cre gene and producing the site-specific recombination.

24. A strain of yeast transformed with the following DNA sequences:

(i) a DNA sequence comprising a regulatory nucleotide sequence and a cre gene,

(ii) a DNA sequence comprising a first lox site, and

(iii) a DNA sequence comprising a second lox site.

25. A strain of yeast as defined in claim 24, wherein the DNA sequences comprising lox sites are connected by a pre-selected DNA segment.

26. A strain of yeast as defined in claim 25, wherein the cre gene is isolated from bacteriophage P1.

27. A strain of yeast as defined in claim 26, wherein the first and second lox sites are loxP or loxC2.

28. Yeast strain BSY90 having deposit accession number ATCC 20772.

29. Yeast strain pBSY23 having deposit accession number ATCC 20773.

30. A plasmid having a regulatory nucleotide sequence and a cre gene, said plasmid being capable of transforming a eukaryotic cell.

31. A plasmid as defined in claim 30, wherein the cre gene is isolated from bacteriophage P1.

32. A plasmid as defined in claim 30, wherein the plasmid is pBS39 characterized by the restriction enzyme map shown in FIG. 1.

33. A plasmid as defined in claim 30, wherein the plasmid is pBS49 characterized by the restriction enzyme map shown in FIG. 1.

34. A plasmid having at least one lox site, said plasmid being capable of transforming a eukaryotic cell.

35. A plasmid as defined in claim 34, wherein a first lox site and a second lox site are connected by a pre-selected DNA segment.

36. A plasmid as defined in claim 35, wherein the first and second lox sites are LoxP sites.

37. A plasmid as defined in claim 34, wherein the plasmid is pBS44 characterized by the restriction enzyme map shown in FIG. 6, or a derivative thereof carrying a pre-selected DNA segment other than or in addition to the LEU2 gene and located between the first and second lox sites.

38. A plasmid as defined in claim 34 wherein the plasmid is pBS47 characterized by the restriction enzyme map shown in FIG. 6, or a derivative thereof carrying a pre-selected DNA segment other than or in addition to the LEU2 gene and located between the first and second lox sites.

39. A plasmid as defined in claim 34 wherein the plasmid is pBS42 characterized by the restriction enzyme map shown in FIG. 2, or a derivative thereof carrying a pre-selected DNA segment other than or in addition to the LEU2 gene and located between the first and second lox site.

40. A plasmid as defined in claim 34 wherein the plasmid s pBS43 characterized by the restriction enzyme map shown in FIG. 2, or a derivative thereof carrying a pre-selected DNA segment other than or in addition to the LEU2 gene and located between the first and second lox sites.