DNA recombination in eukaryotic cells by the bacteriophage PHIC31 recombination system

• US 8,129,598 B2
• Filed: 11/24/2003
• Issued: 03/06/2012
• Est. Priority Date: 07/23/1999
• Status: Expired due to Term

• 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: Ow, David W., Calendar, Richard, Thomason, Lynn
• Primary Examiner(s): Ketter, James S
• Application Number:
  US10/721,980
• Publication Number:
  US 20050054106A1
• Time in Patent Office: 3,025 Days
• Field of Search: None
• US Class Current: 800/317.3
• 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 plant or yeast eukaryotic cell that comprises

a prokaryotic recombinase polypeptide or a nucleic acid that encodes the prokaryotic recombinase, wherein the recombinase is capable of mediating site-specific recombination in the eukaryotic cell between an attB recombination site and an attP recombination site to form an attL and an attR site;

and wherein the recombinase is not capable of mediating in the eukaryotic cell recombination between the attL site and the attR site, wherein the recombinase is a bacteriophage Φ

C31 integrase; and

only one attP or only one attB recombination site of bacteriophage Φ

C31 integrase integrated in its genome.

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

3. The eukaryotic cell of claim 2, wherein the nucleic acid further comprises a selectable marker.

4. The eukaryotic cell of claim 2, wherein the promoter is an inducible or a repressible promoter.

5. The eukaryotic cell of claim 1, wherein the cell is a yeast cell.

6. The eukaryotic cell of claim 1, wherein the eukaryotic cell is a plant cell.

19. The method of claim 4, wherein the eukaryotic cell comprises a polynucleotide that encodes the recombinase polypeptide.

7. A plant or yeast eukaryotic cell that comprises:

an attP or attB recombination site of bacteriophage Φ

C31 integrase integrated in its genome; and

a non-genomic nucleic acid comprising a heterologous nucleic acid or a transgene, and only one attP site of bacteriophage Φ

C31 integrase if the cell has the genomic attB site or only one attB site of bacteriophage Φ

C31 integrase if the cell has the genomic attP site;

wherein the eukaryotic cell further comprises a Φ

C31 integrase polypeptide.

8. The eukaryotic cell of claim 7, wherein the non-genomic nucleic acid comprises the transgene.

9. The eukaryotic cell of claim 7, wherein the eukaryotic cell comprises a nucleic acid that comprises a polynucleotide that encodes the Φ

C31 integrase polypeptide.

10. The eukaryotic cell of claim 9, wherein the nucleic acid further comprises a selectable marker.

11. The eukaryotic cell of claim 9, wherein the nucleic acid further comprises an inducible promoter which controls expression of the Φ

C31 integrase-encoding polynucleotide in the cell.

12. The eukaryotic cell of claim 7, wherein the plant is a dicot or a monocot.

20. A plant regenerated from a plant eucaryotic cell of claim 7.

13. A eucaryotic somatic cell in culture comprising:

a prokaryotic recombinase polypeptide or a nucleic acid that encodes the prokaryotic recombinase, wherein the recombinase is capable of mediating site-specific recombination in the eukaryotic cell between an attB recombination site and an attP recombination site to form an attL and an attR site, and is not capable of mediating in the eukaryotic cell recombination between the attL site and the attR site;

the attP or attB recombination site integrated in its genome;

a non-genomic nucleic acid comprising a transgene or a heterologous nucleic acid and only one attP site if the cell has the genomic attB site or only one attP site if the cell has the genomic attB site;

wherein the recombinase is a bacteriophage Φ

C31 integrase and the attP and attB sites are bacteriophage Φ

C31 integrase recombination sites.

14. A method for obtaining site-specific recombination in a eukaryotic cell, the method comprising:

providing a eukaryotic cell that comprises an attB recombination site or an attP recombination site of bacteriophage Φ

C31 integrase integrated in its genome and a non-genomic nucleic acid comprising a transgene or a heterologous nucleic acid and only one attP site if the cell has the genomic attB site or only one attP site if the cell has the genomic attB site;

contacting the attB and the attP recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites, thereby forming an attR and an attL recombination site;

wherein the recombinase polypeptide can mediate site-specific recombination between the attB and attP recombination sites, but cannot mediate recombination between the attR and attL recombination sites;

wherein the recombinase is-a bacteriophage Φ

C31 integrase.

15. The method of claim 14, wherein the eukaryotic cell is selected from the group consisting of a yeast cell, a fungal cell, a plant cell, an insect cell and an animal cell.

16. The 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 of bacteriophage Φ

C31 integrase, wherein the attB recombination site and the attP recombination site are present on a single nucleic acid molecule;

contacting the attB and the attP recombination sites with a prokaryotic recombinase polypeptide, resulting in recombination between the recombination sites, thereby forming an attR and an attL recombination site;

wherein the recombinase polypeptide can mediate site-specific recombination between the attB and attP recombination sites, but cannot mediate recombination between the attR and attL recombination sites; and

wherein the recombinase is-a bacteriophage Φ

C31 integrase.

17. The method of claim 16, wherein the attB recombination site and the attP recombination site are in a direct orientation and the recombination results in excision of the portion of the nucleic acid molecule that lies between the attB and attP recombination sites.

18. The method of claim 16, wherein the attB recombination site and the attP recombination site are in an inverted orientation and the recombination results in inversion of the portion of the nucleic acid molecule that lies between the attB and attP recombination sites.