Novel FRT recombination sites and methods of use

• US 20080047031A1
• Filed: 07/14/2006
• Published: 02/21/2008
• 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: Lyznik, Leszek, Bidney, Dennis, Tao, Yumin, Gordon-Kamm, William
• Granted Patent: US 7,736,897 B2
• Time in Patent Office: Days
• US Class Current: 800/278
• 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, the first FRT recombination site comprises a spacer sequence selected from the group consisting of SEQ ID NOS:

1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.

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

21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

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 aft 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 aft site is selected from the group consisting of a wild type aft site, and a modified att site.

6. The isolated polynucleotide of claim 4, wherein the second recombination site comprises a polynucleotide selected from the group consisting SEQ ID NO:

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38.

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.

9. A cell comprising the polynucleotide of claim 1.

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, tobacco, Arabidopsis, or cotton.

14. A plant comprising the cell of claim 10.

15. A seed having stably integrated into its genome the polynucleotide of claim 1.

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.

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.

20. 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 selected from the group consisting of:

i) an FRT recombination site comprising a spacer region selected from the group consisting of SEQ ID NOS;

1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18; and

,

ii) an FRT recombination site selected from the group consisting of 21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38;

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.

21. The method of claim 20 wherein the first DNA and the second DNA are provided on a single polynucleotide molecule.

22. The method of claim 20 wherein the first site-specific recombination site and the second site-specific recombination sites are corresponding sites.

23. The method of claim 20 wherein the first site-specific recombination site and the second site-specific recombination sites are dissimilar sites.

24. The method of claim 21 wherein the first site-specific recombination site and the second site-specific recombination site are directly oriented relative to each other.

25. The method of claim 24 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.

26. The method of claim 25 wherein excision of the first polynucleotide of interest activates expression of a second polynucleotide of interest.

27. The method of claim 21 wherein the first site-specific recombination site and the second site-specific recombination site are in the opposite orientation relative to each other.

28. The method of claim 27 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.

29. The method of claim 28 wherein inversion of the first polynucleotide of interest activates expression of the first polynucleotide of interest.

30. The method of claim 28 wherein inversion of the first polynucleotide of interest activates expression of a second polynucleotide of interest.

31. The method of claim 20 wherein the first DNA is provided on a first polynucleotide and the second DNA is provided on a second polynucleotide, wherein the first and the second polynucleotides are separate molecules.

32. The method of claim 31, wherein the second polynucleotide is a circular molecule.

33. The method of claim 31 wherein the second polynucleotide further comprises a polynucleotide of interest

34. The method of claim 20 wherein the second site-specific recombination site is a modified FRT recombination site selected from the group consisting of SEQ ID NOS:

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38.

35. The method of claim 20 wherein the site-specific recombination occurs in vivo.

36. The method of claim 35 wherein the site-specific recombination occurs in a eukaryotic cell.

37. The method of claim 36 wherein the eukaryotic cell is a plant cell.

38. The method of claim 37 wherein the plant cell is from a plant selected from the group consisting of maize, rice, wheat, barley, millet, sorghum, rye, soybean, alfalfa, canola, Arabidopsis, tobacco, sunflower, cotton, and safflower.

39. The method of claim 20, wherein providing the first recombination site, the second recombination site, or the recombinase comprises transformation.

40. The method of claim 20, wherein providing the first recombination site, the second recombination site, or the recombinase comprises sexual breeding.

41. The method of claim 20, wherein the first recombination site is stably incorporated in the genome of a cell.

42. The method of claim 20 comprising:

a) providing a polynucleotide comprising the first FRT recombination site and the second recombination site, wherein the second recombination site is a FRT site;

b) providing a FLP recombinase under conditions such that the FLP recombinase implements a recombinase-mediated excision event; and

,

c) determining excision efficiency of the first and the second FRT recombination sites.

43. The method of claim 42, wherein the first and the second FRT recombination sites are corresponding recombination sites.

44. The method of claim 20 comprising

a) providing the first FRT recombination site;

b) providing the second recombination site, wherein the second recombination site is a FRT site that is dissimilar to the first FRT recombination site;

c) providing a FLP recombinase under conditions such that the FLP recombinase implements a recombination event; and

,

d) assaying for a recombination event to determine if the first and the second recombination site are non-recombinogenic.

45. The method of claim 20 comprising:

a) providing a target site comprising the first FRT recombination site;

b) providing a transfer cassette comprising the second recombination site and a polynucleotide of interest, wherein the first and the second recombination sites are recombinogenic with respect to one another; and

,

c) providing the recombinase wherein the recombinase recognizes and implements recombination at the first and the second recombination sites, and the polynucleotide of interest is inserted at the target site.

46. The method of claim 45, wherein the target site is stably incorporated into the genome of a cell.

47. The method of claim 20 comprising:

a) providing a target site comprising the first FRT site and the second recombination site, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with respect to one another;

b) providing a transfer cassette comprising a polynucleotide of interest, wherein the polynucleotide of interest is flanked by the first and the second recombination sites, and,

c) providing the recombinase, wherein the recombinase recognizes and implements recombination at the first and the second recombination sites, and the polynucleotide of interest is inserted at the target site.

48. The method of claim 47, wherein the target site comprises a second polynucleotide of interest flanked by the first and the second recombination site.

49. The method of claim 47, wherein the target site is stably incorporated into the genome of a cell.

50. The method of claim 47, comprising:

a) the target site operably linked to a first and a second promoter, wherein the first promoter and the second promoter are in a convergent orientation;

b) the transfer cassette comprising in the following order, the first functional recombination site, a first polynucleotide of interest orientated in the 5′

to 3′

direction, a second polynucleotide of interest oriented in the 3′

to 5′

direction, and the second functional recombination site;

c) providing the recombinase wherein insertion of the transfer cassette at the target site results in the first polynucleotide of interest being operably linked to the first convergent promoter and the second polynucleotide of interest being operably linked to the second convergent promoter.

51. The method of claim 20 comprising:

a) providing a target site comprising the first FRT site and the second recombination site, wherein the first and the second recombination sites are dissimilar and non-recombinogenic with respect to one another;

b) providing a first transfer cassette comprising in the following order 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 polynucleotide of interest, and the first, the second, and the third recombination sites are dissimilar and non-recombinogenic with respect to one another;

c) providing a first recombinase, wherein the first recombinase recognizes and implements recombination at the first and the second recombination sites;

d) providing a second transfer cassette comprising the second and the third recombination sites, wherein the second and the third recombination sites of the second transfer cassette flank a second polynucleotide of interest; and

,

e) providing a second recombinase, wherein the second recombinase recognizes and implements recombination at the second and third recombination sites, whereby the first and the second transfer cassettes are integrated at the target site.

52. The method of claim 20 comprising:

a) providing a target site comprising in the following order at least the first FRT recombination site, the second recombination site, and a third recombination site;

wherein the first, the second, and the third recombination sites are dissimilar and non-recombinogenic with respect to one another;

b) providing a first transfer cassette comprising a first polynucleotide of interest flanked by the first and the second recombination sites;

c) providing a first recombinase, wherein the first recombinase recognizes and implements recombination at the first and the second recombination sites;

d) providing a second transfer cassette comprising a second polynucleotide of interest flanked by at least the second and the third recombination sites; and

,

e) providing a second recombinase, wherein the second recombinase recognizes and implements recombination at the second and third recombination sites, whereby the first and the second transfer cassettes are integrated at the target site.

53. An isolated library comprising a population of plasmids wherein each plasmid in the population comprises a first common selectable marker;

and each plasmid in the population comprises a member of a population of modified FRT recombination sites, wherein each member of the population of modified FRT recombination sites comprises a spacer region comprising at least one nucleotide alteration in SEQ ID NO;

43, wherein the population of plasmids comprises at least about 100 distinct members of the plasmid population.

54. The isolated library of claim 53, wherein the modified FRT recombination sites of the population are recombinogenic.

55. The isolated library of claim 53, wherein the modified FRT recombination sites do not include modified FRT recombination sites with a spacer region selected from the group consisting of SEQ ID NOS:

4, 44, 55, 56, 57, 58, 59, 60, 61, 62, 63, and 64.

56. The isolated library of claim 53, wherein the population of plasmids comprises at least one modified recombinogenic FRT site selected from the group consisting of:

a) a polynucleotide comprising a spacer region selected from the group consisting of SEQ ID NOS;

1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18, and;

b) a modified recombinogenic FRT site selected from the group consisting of SEQ ID NO;

21, 22, 23, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, and 38.