FRT recombination sites and methods of use

• US 8,318,493 B2
• Filed: 04/08/2010
• Issued: 11/27/2012
• 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:
  US12/756,211
• Publication Number:
  US 20100192263A1
• Time in Patent Office: 964 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. An isolated polynucleotide comprising a nucleotide sequence comprising at least a first FRT recombination site, wherein the first FRT recombination site comprises the spacer sequence of SEQ ID NO:

6.

2. The polynucleotide of claim 1, wherein the FRT recombination site comprises SEQ ID NO:

26.

3. The isolated polynucleotide of claim 1, wherein the polynucleotide further comprises a second recombination site.

4. The isolated polynucleotide of claim 3, wherein the second recombination site is selected from the group consisting of a FRT site, a LOX site, and an att site.

5. The isolated polynucleotide of claim 4, wherein the FRT site is selected from the group consisting of a wild type FRT site or a modified FRT site, and the LOX site is selected from the group consisting of a wild type LOX site, and a modified LOX site, and the att site is selected from the group consisting of a wild type att site, and a modified att site.

6. The isolated polynucleotide of claim 4, wherein the second recombination site comprises the polynucleotide of SEQ ID NO:

4.

7. The isolated polynucleotide of claim 3, wherein the second recombination site is dissimilar and non-recombinogenic with respect to the first FRT recombination site.

8. The isolated polynucleotide of claim 3, wherein the first FRT recombination site and the second recombination site are corresponding recombination sites.

18. The isolated polynucleotide of claim 3, wherein the polynucleotide comprises a first convergent promoter, the first recombination site, the second recombination site, and a second convergent promoter.

19. The isolated polynucleotide of claim 18, wherein the polynucleotide comprises the first convergent promoter, the first recombination site, a first polynucleotide sequence of interest operably linked to the first convergent promoter, a second polynucleotide of interest operably linked to the second convergent promoter, the second recombination site, and the second convergent promoter.

9. A cell comprising a heterologous polynucleotide comprising a nucleotide sequence comprising at least a first FRT recombination site, the first FRT recombination site comprises the spacer sequence of SEQ ID NO:

6.

10. The cell of claim 9, wherein the cell is from a plant.

11. The cell of claim 9, wherein the polynucleotide is stably integrated into the genome of the cell.

12. The cell of claim 10, wherein the cell is from a monocotyledonous plant or a dicotyledonous plant.

13. The cell of claim 12, wherein the plant cell is from maize, barley, millet, wheat, rice, sorghum, rye, soybean, canola, alfalfa, sunflower, safflower, sugarcane, tobacco, Arabidopsis, or cotton.

14. A plant comprising the cell of claim 10.

16. The cell of claim 9, wherein the cell further has stably incorporated into its genome a nucleic acid molecule encoding a FLP recombinase.

17. The cell of claim 16, wherein the nucleic acid molecule encodes a biologically active variant of the FLP recombinase.

20. The cell of claim 9, wherein said FRT recombination site comprises SEQ ID NO:

26.

21. The cell of claim 9, wherein said polynucleotide further comprises a second recombination site.

15. A seed having stably integrated into its genome a heterologous polynucleotide comprising a nucleotide sequence comprising at least a first FRT recombination site, wherein the first FRT recombination site comprises the spacer sequence of SEQ ID NO:

6.