DNA recombination in eukaryotic cells by the bacteriophage PHIC31 recombination system

• US 20050054106A1
• Filed: 11/24/2003
• Published: 03/10/2005
• Est. Priority Date: 07/23/1999
• Status: Active Grant

• Current Assignee: United States Department Of Agriculture, Regents of the University of California (University of California)
• Original Assignee: United States Department Of Agriculture, Regents of the University of California (University of California)
• Inventors: Thomason, Lynn, Ow, David W., Calendar, Richard
• Granted Patent: US 8,129,598 B2
• Time in Patent Office: Days
• US Class Current: 435/456
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/8201 Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 2830/00 Vector systems having a special element relevant for transcription
• CPC Class Codes: C12N 2830/005 repressible enhancer/promoter combination, e.g. KRAB
• CPC Class Codes: C12N 2830/55 from bacteria
• CPC Class Codes: C12N 2830/706 S. pombe
• CPC Class Codes: C12N 2840/20 translation of more than one cistron

Abstract

This invention provides methods for obtaining specific and stable integration of nucleic acids into eukaryotic cells. The invention makes use of site-specific recombination systems that use prokaryotic recombinase polypeptides, such as the ΦC31 integrase, that can mediate recombination between the recombination sites, but not between hybrid recombination sites that are formed upon the recombination. Thus, the recombination is irreversible in the absence of additional factors. Eukaryotic cells that contain the recombinase polypeptides, or genes that encode the recombinases, are also provided.

Claims

1. A eukaryotic cell that comprises a prokaryotic recombinase polypeptide or a nucleic acid that encodes a prokaryotic recombinase, wherein the recombinase can mediate site-specific recombination between a first recombination site and a second recombination site that can serve as a substrate for recombination with the first recombination site, but in the absence of an additional factor that is not present in the eukaryotic cell cannot mediate recombination between two hybrid recombinase recombination sites that are formed upon recombination between the first recombination site and the second recombination site.

2. The eukaryotic cell of claim 1, wherein the recombinase is selected from the group consisting of a bacteriophage Φ

C31 integrase, a coliphage P4 recombinase, a Listeria phage recombinase, a bacteriophage R4 Sre recombinase, a CisA recombinase, an XisF recombinase, and a transposon Tn4451 TnpX recombinase.

3. The eukaryotic cell of claim 1, wherein the recombinase is a bacteriophage Φ

C31 integrase

4. The eukaryotic cell of claim 1, wherein the first recombination site is an attB site and the second recombination site is an attP site.

5. The eukaryotic cell of claim 1, wherein the cell further comprises a first recombinase recombination site.

6. The eukaryotic cell of claim 1, wherein the cell comprises a nucleic acid that comprises a coding sequence for an recombinase polypeptide, which coding sequence is operably linked to a promoter that mediates expression of the recombinase-encoding polynucleotide in the eukaryotic cell.

7. The eukaryotic cell of claim 6, wherein the nucleic acid further comprises a selectable marker.

8. The eukaryotic cell of claim 6, wherein the promoter is an inducible or a repressible promoter.

9. The eukaryotic cell of claim 8, wherein the nucleic acid is the plasmid pLT43.

10. The eukaryotic cell of claim 1, wherein the eukaryotic cell is selected from the group consisting of an animal cell, a plant cell, a yeast cell, an insect cell and a fungal cell.

11. The eukaryotic cell of claim 10, wherein the eukaryotic cell is a mammalian cell.

12. The eukaryotic cell of claim 10, wherein the eukaryotic cell is present in a multicellular organism.

13-31. -31. (canceled).

32. A nucleic acid that comprises a polynucleotide sequence that encodes a bacterial recombinase polypeptide operably linked to a promoter that functions in a eukaryotic cell, wherein the recombinase polypeptide cannot mediate recombination between two hybrid recombination sites that are formed upon recombination between a first recombination site and a second recombination site in the absence of an additional factor.

33. The nucleic acid of claim 32, wherein the nucleic acid further comprises at least one recombination site that is recognized by the recombinase polypeptide.

34. The nucleic acid of claim 32, wherein the nucleic acid comprises a plasmid vector.

35. The nucleic acid of claim 34, wherein the vector is pLT43.

36. A eukaryotic cell that comprises a polynucleotide that comprises a first bacteriophage Φ

C31 recombination site.

37. The eukaryotic cell of claim 36, wherein the recombination site is selected from the group consisting of attP and attB.

38. The eukaryotic cell of claim 36, wherein the eukaryotic cell further comprises a second polynucleotide that comprises a second Φ

C31 recombination site that undergoes recombination with the first Φ

C31 recombination site when contacted with a φ

C31 integrase polypeptide.

39. The eukaryotic cell of claim 38, wherein:

the first recombination site is attB and the second recombination site is attP;

or

the first recombination site is attP and the second recombination site is attB.

40. The eukaryotic cell of claim 38, wherein the second polynucleotide further comprises a transgene.

41. The eukaryotic cell of claim 38, wherein the second polynucleotide further comprises a selectable marker.

42. The eukaryotic cell of claim 36, wherein the eukaryotic cell further comprises a Φ

C31 integrase polypeptide

43. The eukaryotic cell of claim 36, wherein the eukaryotic cell further comprises a nucleic acid that comprises a polynucleotide that encodes a Φ

C31 integrase polypeptide.

44. The eukaryotic cell of claim 43, wherein the nucleic acid further comprises a selectable marker.

45. The eukaryotic cell of claim 43, wherein the nucleic acid further comprises a promoter which results in expression of the Φ

C31 integrase-encoding polynucleotide in the cell.

46. The eukaryotic cell of claim 45, wherein the promoter is an inducible promoter.

47. The eukaryotic cell of claim 36, wherein the eukaryotic cell is selected from the group consisting of a yeast cell, a fungal cell, a plant cell, and an animal cell.