Resolution of complex integration patterns to obtain single copy transgenes

• US 6,114,600 A
• Filed: 10/19/1998
• Issued: 09/05/2000
• Est. Priority Date: 10/31/1997
• Status: Expired due to Fees

• Current Assignee: United States Department Of Health And Human Services (Government of the United States of America), 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: Srivastava, Vibha, Ow, David W.
• Primary Examiner(s): Smith, Lynette R. F.
• Assistant Examiner(s): Mehta, Ashwin D.
• Application Number:
  US09/175,219
• Time in Patent Office: 687 Days
• Field of Search: 435/69.1, 435/320.1, 435/410, 435/419, 435/468, 435/469, 435/470, 800/278, 800/288, 800/295, 800/298, 800/320.3, 800/292, 800/293, 800/294, 800/320
• US Class Current: 800/278
• 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 15/8207 by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/90 Stable introduction of foreign DNA into chromosome

Abstract

The present invention provides methods for producing a transgenic cell having a stably integrated, single copy of an exogenous polynucleotide sequence. The method, which resolves repeated insertions of the introduced polynucleotide sequence into a single copy, involves introducing into a genetic locus of a cell a polynucleotide sequence flanked on each end by a recombination site. The recombination sites are oriented such that contact with a recombinase would not result in deletion of the polynucleotide sequence from the construct. The multiple, tandem copies of the introduced polynucleotide locus are then contacted with a recombinase that catalyzes recombination among the recombination sites. As a result of this method, the multiple, tandem copies are resolved to a single copy.

Claims

1. A method of resolving multiple copies of a construct integrated at a locus in a plant cell to a single copy of said construct at said locus, the method comprising:

introducing into the cell a construct comprising an exogenous polynucleotide sequence flank by a first pair of recombination sites oriented such that contact with a recombinase would not result in deletion of the polynucleotide sequence from the construct to obtain a cell which comprises multiple copies of the construct integrated at a locus of a cell chromosome; and

contacting the locus with a recombinase that catalyzes recombination among the recombination sites of the multiple copies at said locus;

wherein the recombination results in resolution of the multiple copies of the introduced construct into a single copy of the construct integrated at said locus.

2. The method of claim 1, wherein the first pair of recombination sites are oriented in the opposite orientation.

3. The method of claim 1, wherein the recombinase is a Cre polypeptide and the recombination sites are lox sites.

4. The method of claim 1, wherein the recombinase and recombination sites are selected from the group consisting of FLP/FRT, and R/RS.

5. The method of claim 1, wherein a selectable marker is located between the first pair of recombination sites.

6. The method of claim 5, wherein said selectable marker is flanked by a second pair of recombination sites, wherein the second pair of recombination sites do not recombine with the first pair of recombination sites and are oriented such that said contacting with said recombinase results in deletion of said selectable marker and one of said second pair of recombination sites from the construct such that said construct integrated at said locus comprises said exogenous polynucleotide flanked by said first pair of recombination sites and the other of said second pair of recombination sites.

7. The method of claim 6, wherein the first pair of recombination sites and the second pair of recombination sites are different species of lox sites.

8. The method of claim 1, wherein the recombinase is encoded by a recombinase gene which is present in the cell.

9. The method of claim 8, wherein the recombinase gene is under the control of an inducible promoter.

10. The method of claim 8, wherein the recombinase gene is introduced into the cell which comprises multiple copies of the introduced construct by crossing with a cell which comprises the recombinase gene.

11. The method of claim 8, wherein the recombinase gene is introduced into the cell simultaneously with the introduction of the introduced construct comprising the exogenous polynucleotide sequence.

12. The method of claim 11, wherein the recombinase gene is present on a polynucleotide which comprises the introduced construct comprising the exogenous polynucleotide sequence.

13. The method of claim 1, wherein the cell comprises a multicellular organism.

14. The method of claim 13, wherein the multicellular organism is a plant.

15. The method of claim 14, wherein the plant is a monocot.

16. The method of claim 15, wherein the monocot is a wheat plant.

17. The method of claim 1, wherein the introduced construct is introduced into the cell by a method selected from the group consisting of particle-mediated DNA transfer, Agrobacterium-mediated transfer, microinjection, electroporation and polyethylene glycol-mediated (PEG) transfer.

18. The method of claim 1, wherein the method further comprises introducing into the cell a second exogenous polynucleotide sequence which comprises a second polynucleotide sequence and a recombination site, wherein the recombination site can recombine with a previously introduced recombination site, resulting in integration of the second polynucleotide sequence at the previously introduced recombination site.