Modified FRT Recombination Site Libraries and Methods of Use

• US 20100173801A1
• Filed: 02/16/2010
• Published: 07/08/2010
• 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 A., TAO, YUMIN, BIDNEY, DENNIS L., GORDON-KAMM, WILLIAM J.
• Granted Patent: US 9,234,194 B2
• Time in Patent Office: Days
• US Class Current: 506/10
• 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. Kits comprising the library populations of FRT sites are also provided, as are methods to make a library of modified FRT recombination sites. The recombinogenic modified FRT recombination sites can be employed in a variety of methods for targeted recombination of polynucleotides of interest.

Claims

1. 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.

2. The isolated library of claim 1, wherein the modified FRT recombination sites of the population are recombinogenic.

3. The isolated library of claim 1, 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.

4. The isolated library of claim 1, 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.

5. A kit comprising the population of plasmids of claim 1, further comprising a second population of plasmids wherein each plasmid in the second population comprises a second common selectable marker, wherein the first and the second selectable markers are distinct;

and, each plasmid in the second population comprises a member of the population of modified FRT recombination sites.

6. The kit of claim 5, wherein the kit further comprises a FLP recombinase or a polynucleotide encoding the FLP recombinase.

7. The kit of claim 5, wherein at least one member of the first population, the second population, or both populations of modified FRT recombination sites comprises:

a) 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 NOS;

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

8. The kit of claim 5 wherein each member of the first and the second population of modified FRT recombination site is recombinogenic.

9. A method for generating a library of molecules comprising

a) providing 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; and

,

b) contacting the population of modified FRT recombination sites with a population of plasmids having a common selectable marker under conditions for the insertion of the population of modified FRT recombination sites into the population of plasmids, such that each of the plasmids of the population comprises a single member of the population of modified FRT recombination sites to generate a library of molecules.

15. The method of claim 9, wherein the first population of plasmids and the second population of plasmids are combined in an equimolar ratio.

10. A method to select a recombinogenic modified FRT recombination site comprising:

a) providing a first population of plasmids wherein each plasmid in the first population comprises a common first selectable marker; and

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

43;

b) providing a second population of plasmids wherein each plasmid in the second population comprises a common second selectable marker, wherein the first and the second selectable markers are distinct; and

, each plasmid in the second population comprises a member of the population of modified FRT recombination sites;

c) combining the first population of plasmids with the second population of plasmids in the presence of a FLP recombinase under conditions where site-specific recombination can occur; and

,

d) selecting for a co-integrant plasmid comprising the first and the second selectable marker, the co-integrate plasmid comprising the modified FRT recombination site.

11. The method of claim 10, wherein the co-integrant plasmid comprises a functional modified FRT recombination site.

12. The method of claim 10, further comprising isolating the co-integrant plasmid.

13. The method of claim 10, further comprising characterizing the modified FRT recombination site of the co-integrant plasmid.

14. The method of claim 13, wherein characterizing the modified FRT recombination site comprises a method selected from the group consisting of determining excision efficiency, determining recombination specificity, and sequencing the modified FRT recombination site.