DNA recombination in eukaryotic cells by the bacteriophage PHIC31 recombination system

• US 6,746,870 B1
• Filed: 07/21/2000
• Issued: 06/08/2004
• Est. Priority Date: 07/23/1999
• Status: Expired due to Term

• Current Assignee: of Agriculture United States As Represented By The Secretary, 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: Ow, David W., Calendar, Richard, Thomason, Lynn
• Primary Examiner(s): Yucel, Remy
• Assistant Examiner(s): KATCHEVES, KONSTANTINA T
• Application Number:
  US09/620,800
• Time in Patent Office: 1,418 Days
• Field of Search: 435/477, 435/252.35, 435/320.1, 435/471
• US Class Current: 435/477
• 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 method for obtaining site-specific recombination in a eukaryotic cell, the method comprising:

providing a eukaryotic cell that comprises an attB recombination site and an attP recombination site, which attP recombination site can serve as a substrate for recombination with the attB recombination site;

contacting the attB and the attP recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the attB and attP recombination sites;

thereby forming an attL site and an attR site;

wherein the recombinase polypeptide can mediate site-specific recombination between the attB and attP recombination sites, but cannot mediate recombination between the attL site and the attR site formed from recombination between the attB recombination site and the attP recombination site in the absence of an additional factor that is not present in the eukaryotic cell; and

wherein the eukaryotic cell comprises a polynucleotide that encodes the recombinase polypeptide.

2. The method of claim 1, wherein the recombinase-encoding polynucleotide is operably linked to a promoter which mediates expression of the polynucleotide in the eukaryotic cell.

3. The method of claim 2, wherein the promoter is an inducible or a repressible promoter.

4. The method of claim 3, wherein the promoter is a Pmnt promoter.

5. A method for obtaining a eukaryotic cell having a stably integrated transgene, the method comprising:

introducing a nucleic acid into a eukaryotic cell that comprises a first attB or attP recombination site, wherein the nucleic acid comprises a transgene and a second recombination site which is an attP site, when the first site is attB site, or an attB site, when the first site is attP site, which second recombination site can serve as a substrate for recombination with the first recombination site; and

contacting the first and the second recombination sites with a prokaryotic recombinase polypeptide, wherein the recombinase polypeptide catalyzes recombination between the first and second recombination sites, resulting in integration of the nucleic acid at the first recombination site, thereby forming an attL site and an attR site at each end of the nucleic acid;

wherein the recombinase polypeptide can mediate site-specific recombination between the first and second recombination sites, but cannot mediate recombination between the attL and attR sites in the absence of an additional factor that is not present in the eukaryotic cell.

6. The method of claim 5, wherein the recombinase polypeptide 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.

7. The method of claim 6, wherein the recombinase is a Φ

C31 integrase.

8. The method of claim 5, wherein the recombinase polypeptide is introduced into the eukaryotic cell by expression of a polynucleotide that encodes the recombinase polypeptide.

9. The method of claim 8, wherein the polynucleotide that encodes the recombinase polypeptide is operably linked to a promoter that functions in the eukaryotic cell.

10. The method of claim 9, wherein the promoter is an inducible or a repressible promoter.