Modified FRT recombination site libraries and methods of use
First Claim
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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.
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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.
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Citations
15 Claims
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
- 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;
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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. - View Dependent Claims (15)
- 43; and
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10. A method to select a recombinogenic modified FRT recombination site comprising:
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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. - View Dependent Claims (11, 12, 13, 14)
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Specification