Method for selecting recombinase variants with altered specificity

• US 6,890,726 B1
• Filed: 04/06/2000
• Issued: 05/10/2005
• Est. Priority Date: 04/06/1999
• Status: Expired due to Fees

• Current Assignee: Oklahoma Medical Research Foundation
• Original Assignee: Oklahoma Medical Research Foundation
• Inventors: Rufer, Andreas Walter, Sauer, Brian Lee
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Lambertson, David A.
• Application Number:
  US09/544,045
• Time in Patent Office: 1,860 Days
• Field of Search: 435/7.4, 435/6, 435/455, 536/23.1
• US Class Current: 435/7.4
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries

Abstract

Disclosed are variants of Cre recombinase that have broadened specificity for the site of recombination. Specifically, the disclosed variants mediate recombination between sequences other than the loxP sequence and other lox site sequences on which wild type Cre recombinase is active. In general, the disclosed Cre variants mediate efficient recombination between lox sites that wild type Cre can act on (referred to as wild type lox sites), between variant lox sites not efficiently utilized by wild type Cre (referred to as variant lox sites), and between a wild type lox site and a variant lox site. Also disclosed are methods or recombining nucleic acids using the disclosed Cre variants. For example, the disclosed Cre variants can be used in any method or technique where Cre recombinase (or other, similar recombinases such as FLP) can be used. In addition, the disclosed Cre variants allow different alternative recombinations to be performed since the Cre variants allow much more efficient recombination between wild type lox sites and variant lox sites. Control of such alternative recombination can be used to accomplish more sophisticated sequential recombinations to achieve results not possible with wild type Cre recombinase.

Claims

1. A method of identifying variant recombinases that mediate recombination at variant recombination sites, the method comprising,

(a) bringing into contact

a mutant recombinase,

a first nucleic acid sequence comprising a first reporter gene and first and second recombination sites, wherein the first and second recombination sites are variant recombination sites, and

a second nucleic acid sequence comprising a second reporter gene and third and fourth recombination sites, wherein the third and fourth recombination sites can be recombined by a non-mutant recombinase,

(b) determining if recombination occurs between the first and second recombination sites, and determining if recombination occurs between the third and fourth recombination sites,

wherein recombination between the first and second recombination sites indicates that the mutant recombinase is a variant recombinase that mediates recombination at variant recombination sites,

wherein recombination between the third and fourth recombination sites indicates that the mutant recombinase retains the ability to mediate recombination at non-variant recombination sites.

2. The method of claim 1 wherein recombination frequency between the first and second recombination sites mediated by a non-mutant recombinase is significantly reduced.

3. The method of claim 1 wherein the first and second nucleic acid constructs are on the same nucleic acid construct.

4. The method of claim 1 wherein the recombination sites comprise recognition sequences and compatibility sequences,

wherein the recognition sequences of the first and second recombination sites differ from the recognition sequences of the third and fourth recombination sties,

wherein the compatibility sequences of the first and second recombination sites are sufficiently similar to allow recombination between the first and second recombination sites, and wherein the compatibility sequences of the third and fourth recombination sites are sufficiently similar to allow recombination between the third and fourth recombination sites, and

wherein the compatibility sequences of the first and second recombination sites differ from the compatibility sequences of the third and fourth recombination sites such that neither the first nor the second recombination site can be recombined with either the third or the fourth recombination site.

5. The method of claim 1 or 4 wherein the first and second recombination sites have identical sequences, and wherein the third and fourth recombination sites have identical sequences.

6. The method of claim 1 wherein the first nucleic acid sequence is a first nucleic acid construct and the second nucleic acid sequence is on a second nucleic acid construct.

7. The method of claim 6 wherein the first nucleic acid construct is an extrachromosomal vector and the second nucleic acid construct is in the genome of a host cell.

8. The method of claim 1 wherein recombination between the first and second recombination sites alters the expression of the first reporter gene, wherein recombination between the first and second recombination sites is determined by determining if expression of the first reporter gene is altered, and

wherein recombination between the third and fourth recombination sites alters the expression of the second reporter gene, wherein recombination between the third and fourth recombination sites is determined by determining if expression of the second reporter gene is altered.

9. The method of claim 8 wherein recombination between the first and second recombination sites allows the first reporter gene to be expressed.

10. The method of claim 9 wherein the first nucleic acid sequence further comprises a spacer sequence flanked by the first and second recombination sites, wherein the spacer sequence interrupts the first reporter gene such that the first reporter gene is not expressed, wherein recombination of the first and second recombination sites excises the spacer sequence which allows the first reporter gene to be expressed.

11. The method of claim 9 wherein a portion of the first reporter gene is inverted, wherein the inverted portion of the first reporter gene is flanked by the first and second recombination sites, wherein recombination of the first and second recombination sites inverts the inverted portion of the first reporter gene which allows the first reporter gene to be expressed.

12. The method of claim 8 wherein recombination between the first and second recombination sites prevents expression of the first reporter gene.

13. The method of claim 12 wherein the first reporter gene is flanked by the first and second recombination sites, wherein recombination of the first and second recombination sites excises the first reporter gene which prevents expression of the first reporter gene.

14. The method of claim 12 wherein a portion of the first reporter gene is flanked by the first and second recombination sites, wherein recombination of the first and second recombination sites inverts the flanked portion of the first reporter gene which prevents expression of the first reporter gene.

15. The method of claim 8 wherein recombination between the third and fourth recombination sites prevents expression of the second reporter gene to be expressed.

16. The method of claim 15 wherein the second reporter gene is flanked by the third and fourth recombination sites, wherein recombination of the third and fourth recombination sites excises the second reporter gene which prevents expression of the second reporter gene.

17. The method of claim 15 wherein a portion of the second reporter gene is flanked by the third and fourth recombination sites, wherein recombination of the third and fourth recombination sites inverts the flanked portion of the second reporter gene which prevents expression of the second reporter gene.

18. The method of claim 8 wherein recombination between the third and fourth recombination sites allows the second reporter gene to be expressed.

19. The method of claim 18 wherein a portion of the second reporter gene is inverted, wherein the inverted portion of the second reporter gene is flanked by the third and fourth recombination sites, wherein recombination of the third and fourth recombination sites inverts the inverted portion of the second reporter gene which allows the second reporter gene to be expressed.

20. The method of claim 18 wherein the second nucleic acid sequence further comprises a spacer sequence flanked by the third and fourth recombination sites, wherein the spacer sequence interrupts the second reporter gene such that the second reporter gene is not expressed, wherein recombination of the third and fourth recombination sites excises the spacer sequence which allows the second reporter gene to be expressed.

21. The method of claim 20 wherein the spacer sequence interrupts the second reporter gene such that the second reporter gene is not transcribed.

22. The method of claim 20 wherein the second reporter gene encodes a protein, wherein the spacer sequence interrupts the second reporter gene such that the protein encoded by the second reporter gene is not translated.

23. The method of claim 20 wherein the spacer sequence interrupts the second reporter gene such that the second reporter gene produces an inactive expression product.