Novel FRT Recombination Sites and Methods of Use

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.

1 . 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 comprises:
 i) an FRT recombination site comprising a spacer region comprising SEQ ID NO: 4; or, 
 ii) an FRT recombination site comprising SEQ ID NO: 24; 
   b) providing a second DNA comprising a second site-specific recombination 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.   
     
     
         2 . The method of  claim 1 , wherein the first DNA and the second DNA are provided on a single polynucleotide molecule. 
     
     
         3 . The method of  claim 1 , wherein the first site-specific recombination site and the second site-specific recombination sites are corresponding sites. 
     
     
         4 . The method of  claim 1 , wherein the first site-specific recombination site and the second site-specific recombination sites are dissimilar sites. 
     
     
         5 . The method of  claim 2 , wherein the first site-specific recombination site and the second site-specific recombination site are directly oriented relative to each other. 
     
     
         6 . The method of  claim 5 , 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. 
     
     
         7 . The method of  claim 6 , wherein excision of the first polynucleotide of interest activates expression of a second polynucleotide of interest. 
     
     
         8 . The method of  claim 2 , wherein the first site-specific recombination site and the second site-specific recombination site are in the opposite orientation relative to each other. 
     
     
         9 . The method of  claim 8 , 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. 
     
     
         10 . The method of  claim 9 , wherein inversion of first polynucleotide of interest activates expression of the first polynucleotide of interest. 
     
     
         11 . The method of  claim 9 , wherein inversion of the first polynucleotide of interest activates expression of a second polynucleotide of interest. 
     
     
         12 . The method of  claim 1 , wherein the second site-specific recombination site is a modified FRT recombination site comprising SEQ ID NOS: 4 or 24.