FRT recombination sites and methods of use

• US 8,900,869 B2
• Filed: 10/25/2012
• Issued: 12/02/2014
• Est. Priority Date: 07/18/2005
• Status: Active Grant

• Current Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Original Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Inventors: Tao, Yumin, Bidney, Dennis L., Gordon-Kamm, William J., Lyznik, Leszek A.
• Primary Examiner(s): Zheng, Li
• Application Number:
  US13/660,562
• Publication Number:
  US 20130052739A1
• Time in Patent Office: 768 Days
• Field of Search: None
• US Class Current: 435/419
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries

Abstract

Methods and compositions using populations of randomized modified FRT recombination sites to identify, isolate and/or characterize modified FRT recombination sites are provided. The recombinogenic modified FRT recombination sites can be employed in a variety of methods for targeted recombination of polynucleotides of interest, including methods to recombine polynucleotides, assess promoter activity, directly select transformed organisms, minimize or eliminate expression resulting from random integration into the genome of an organism, such as a plant, remove polynucleotides of interest, combine multiple transfer cassettes, invert or excise a polynucleotide, and identify and/or characterize transcriptional regulating regions are also provided.

Claims

1. A method for producing site-specific recombination of DNA comprising

a) providing a first DNA comprising a first site-specific recombination site, wherein the first site-specific recombination site comprises a polynucleotide comprises:

i) an FRT recombination site comprising a spacer region comprising SEQ ID NO;

1;

or,

ii) an FRT recombination site comprising SEQ ID NO;

21;

b) providing a second DNA comprising a second site-specific recombination site which is recombinogenic with the first site-specific recombination site; and

,

c) providing a site-specific recombinase that catalyzes a site-specific recombination between the first and the second site-specific recombination sites.

2. The method of claim 1, wherein the first DNA and the second DNA are provided on a single polynucleotide molecule.

4. The method of claim 1, wherein the first site-specific recombination site and the second site-specific recombination sites are dissimilar sites.

5. The method of claim 2, wherein the first site-specific recombination site and the second site-specific recombination site are directly oriented relative to each other.

6. The method of claim 5, wherein the first and the second site-specific recombination sites flank a first polynucleotide of interest, whereby providing the site-specific recombinase excises the first polynucleotide of interest.

7. The method of claim 6, wherein excision of the first polynucleotide of interest activates expression of a second polynucleotide of interest.

8. The method of claim 2, wherein the first site-specific recombination site and the second site-specific recombination site are in the opposite orientation relative to each other.

9. The method of claim 8, wherein the first and the second site-specific recombination sites flank a first polynucleotide of interest, whereby providing the site-specific recombinase inverts the first polynucleotide of interest.

10. The method of claim 9, wherein inversion of the first polynucleotide of interest activates expression of the first polynucleotide of interest.

11. The method of claim 9, wherein inversion of the first polynucleotide of interest activates expression of a second polynucleotide of interest.

12. The method of claim 1, wherein the second site-specific recombination site is a modified FRT recombination site comprising SEQ ID NOS:

1 or 21.

3. The method of claim wherein the first site-specific recombination site and the second site-specific recombination sites are corresponding sites.