Modified FRT recombination site libraries and methods of use

• US 9,234,194 B2
• Filed: 02/16/2010
• Issued: 01/12/2016
• 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): BOESEN, CHRISTIAN C
• Application Number:
  US12/706,489
• Publication Number:
  US 20100173801A1
• Time in Patent Office: 2,156 Days
• Field of Search: None
• US Class Current: 1/1
• 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. A composition comprising:

(a) a first DNA library comprising a first population of plasmids wherein each plasmid in the first population comprises a 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 modified FRT recombination sites is at least 80% identical to SEQ ID NO;

39, 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 first population of plasmids comprises at least about 100 distinct members and wherein the modified FRT recombination sites in the at least 100 distinct members of the first plasmid population comprise functional modified FRT recombination sites that are recognized by a FLP recombinase and capable of a recombinase mediated recombination reaction; and

,

(b) a second DNA library comprising a second population of plasmids wherein each plasmid in the second population comprises a second selectable marker, wherein the second selectable marker is distinct from the first selectable marker; and

each plasmid in the second population comprises a member of the population of modified FRT recombination sites, wherein each member of the population of modified FRT recombination sites is at least 80% identical to SEQ ID NO;

39, wherein the second population of plasmids comprises at least about 100 distinct members, and wherein the at least 100 distinct members of the second plasmid population comprises a functional modified FRT recombination site that is recognized by a FLP recombinase and capable of a recombinase mediated recombination reaction.

2. The composition 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.

3. A composition comprising:

(a) a first DNA library comprising a first population of plasmids wherein each plasmid in the first population comprises a 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 modified FRT recombination sites comprises a spacer region and is at least 80% identical to SEQ ID NO;

39, wherein the difference between the modified FRT recombination sites and SEQ ID NO;

39 are found in the spacer region, wherein the first population of plasmids comprises at least about 100 distinct members and wherein the modified FRT recombination sites in the at least 100 distinct members of the first plasmid population comprise functional modified FRT recombination sites that are recognized by a FLP recombinase and capable of a recombinase mediated recombination reaction; and

,

(b) a second DNA library comprising a second population of plasmids wherein each plasmid in the second population comprises a second selectable marker, wherein the second selectable marker is different from the first selectable marker; and

each plasmid in the second population comprises a member of the population of modified FRT recombination sites, wherein the second population of plasmids comprises at least about 100 distinct members, and wherein the at least 100 distinct members of the second plasmid population comprises a functional modified FRT recombination site that is recognized by a FLP recombinase and capable of a recombinase mediated recombination reaction.

4. The composition of claim 3, wherein the spacer region of the modified FRT recombination site comprises at least one nucleotide alteration compared to SEQ ID NO:

43 of SEQ ID NO;

39, wherein SEQ ID NO;

43 is the spacer region and SEQ ID NO;

39 is the native FRT recombination site.