NOVEL FRT RECOMBINATION SITES AND METHODS OF USE

• US 20110047655A1
• Filed: 09/29/2010
• Published: 02/24/2011
• Est. Priority Date: 07/18/2005
• Status: Active Grant

• Current Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Original Assignee: Pioneer Hi Bred International Inc (Corteva, Inc.)
• Inventors: Lyznik, Leszek A., Bidney, Dennis L., Tao, Yumin, Gordon-Kamm, William J.
• Granted Patent: US 8,586,361 B2
• Time in Patent Office: Days
• US Class Current: 800/298
• 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 plant cell having stably incorporated into its genome at least a first and a second transfer cassette, wherein

said first transfer cassette comprises a first nucleotide sequence of interest flanked by a first and a second recombination site, wherein said first and said second recombination sites are dissimilar and non-recombinogenic with respect to one another;

said second transfer cassette comprises a second nucleotide sequence of interest flanked by the second recombination site and a third recombination site, wherein said third recombination site is dissimilar and non-recombinogenic to said first and said second recombination site; and

,

said second recombination site is shared between the first and the second transfer cassette, such that the genome of the plant cell comprises in the following order the first recombination site, the first nucleotide sequence of interest, the second recombination site, the second nucleotide sequence of interest, and the third recombination site,

wherein said first, said second and said third recombination sites are selected from the group consisting of FRT1 (SEQ ID NO;

39), FRT12 (SEQ ID NO;

21), and FRT87 (SEQ ID NO;

24).

2. The plant cell of claim 1, wherein said plant cell is from a monocot.

3. The plant cell of claim 2, wherein said monocot is maize.

4. The plant cell of claim 1, wherein said plant cell is from a dicot.

5. The plant cell of claim 4, wherein said dicot is canola, Brassica, soybean, sunflower, or cotton.

6. The plant cell of claim 1, wherein the first recombination site is FRT1 (SEQ ID NO:

39), the second recombination site is FRT12 (SEQ ID NO;

21),and the third recombination site is FRT87 (SEQ ID NO;

24).

7. The plant cell of claim 1, wherein said plant cell further comprises a LOX site or a biologically active variant of the LOX site stably incorporated into its genome.

8. A plant comprising the plant cell of claim 1.

9. A transformed seed comprising the plant cell of claim 1.

10. A method to combine multiple transfer cassettes at one location in a genome of a plant cell, said method comprising:

a) stably incorporating into the genome of the plant cell, a target site comprising at least a first and a second recombination site, wherein said first and said second recombination sites are dissimilar and non-recombinogenic with respect to one another;

b) introducing into the plant cell a first transfer cassette comprising in the following order at least the first, a third, and the second recombination sites, wherein the first and the third recombination sites of the first transfer cassette flank a first nucleotide sequence of interest, and wherein said third recombination site is dissimilar and non-recombinogenic to said first and said second recombination sites;

c) providing a first recombinase that recognizes and implements recombination at the first and the second non-identical recombination sites.

wherein said first, said second and said third recombination sites are selected from the group consisting of FRT1 (SEQ ID NO;

39), FRT12 (SEQ ID NO;

21). and FRT87 (SEQ ID NO;

24).

11. The method of claim 10, wherein said plant cell is from a monocot.

12. The method of claim 11, wherein said monocot is maize.

13. The method of claim 10, wherein said plant cell is from a dicot.

14. The method of claim 13, wherein said dicot is canola, Brassica, soybean, sunflower, or cotton.

15. The method of claim 10, wherein providing said FLP recombinase comprises genetically transforming said plant cell with one or more expression cassettes comprising a nucleotide sequence encoding said FLP recombinase.

22. The method of claim 10, wherein the first recombination site is FRT1 (SEQ ID NO:

39), the second recombination site is FRT12 (SEQ ID NO;

21),and the third recombination site is FRT87 (SEQ ID NO;

24).

16. A method to combine multiple transfer cassettes at one location in a genome of a plant cell, said method comprising:

a) stably incorporating into the genome of the plant cell, a target site comprising in the following order a first, a second, and a third recombination site, wherein said first, said second and said third recombination sites are dissimilar and non-recombinogenic with respect to one another;

b) introducing into the plant cell a first transfer cassette comprising the first and the second recombination sites, wherein the first and the second recombination sites of the first transfer cassette flank a first nucleotide sequence of interest; and

,

c) providing a FLP recombinase that recognizes and implements recombination at the first and the second recombination sites;

wherein said first, said second and said third recombination sites are selected from the group consisting of FRT1 (SEQ ID NO;

39), FRT12 (SEQ ID NO;

21) and FRT87 (SEQ ID NO;

24).

17. The method of claim 16, wherein said plant cell is from a monocot.

18. The method of claim 17, wherein said monocot is maize.

19. The method of claim 18, wherein said plant cell is from a dicot.

20. The method of claim 19, wherein said dicot is canola, Brassica, soybean, sunflower, or cotton.

21. The method of claim 16, wherein providing said FLP recombinase comprises genetically transforming said plant cell one or more expression cassettes comprising a nucleotide sequence encoding said FLP recombinase.