FLP-FRT Recombination
In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase(Flp)derived from the 2 µm plasmid of baker's yeast Saccharomyces cerevisiae.
The 34bp minimal FRT site sequence has the sequence
for which flippase (Flp) binds to both 13-bp 5'-GAAGTTCCTATTC-3' arms flanking the 8 bp spacer, i.e. the site-specific recombination (region of crossover) in reverse orientation. FRT-mediated cleavage occurs just ahead from the asymmetric 8bp core region (5'tctagaaa3') on the top strand and behind this sequence on the bottom strand. Several variant FRT sites exist, but recombination can usually occur only between two identical FRTs but generally not among non-identical ("heterospecific") FRTs.
Senecoff et al. (1987) investigated how nucleotide substitutions within the FRT affected the efficacy of the FLP-mediated recombination. The authors induced base substitutions in either one or both of the FRT sites and tested the concentration of FLP required to observe site-specific recombinations. Every base substitution was performed on each of the thirteen nucleotides within the FRT site (example G to A, T, and C). First, the authors showed that most mutations within the FRT sequence cause minimal effects if present within only one of the two sites. If mutations occurred within both sites, the efficiency of FLP is dramatically reduced. Second, the authors provided data for which nucleotides are most crucial for the binding of FLP and efficacy of the site-specific recombination. If the first nucleotide in both FRT sites is substituted to a cytosine (G to C), the third nucleotide is substituted for a thymine (A to T), or the seventh nucleotide is substituted for an adenosine (G to A), then the efficacy of the FLP-mediated site-specific recombination is reduced more than 100-fold. While a base substitution of any of the aforementioned nucleotides in only one of the FRT sites led to a ten-fold, ten-fold, and five-fold reduction of efficacy, respectively.
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