FLP-FRT recombination

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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(Flp)derived from the 2µm plasmid of baker's yeast Saccharomyces cerevisiae.

The 34bp minimal FRT site sequence has the sequence

5'GAAGTTCCTATTCtctagaaaGtATAGGAACTTC3'

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.[1] Several variant FRT sites exist, but recombination can usually occur only between two identical FRTs but generally not among non-identical ("heterospecific") FRTs.[2][3]

Many available constructs include an additional arm sequences (5'-GAAGTTCCTATTCC-3') one base pair away from the upstream element and in the same orientation:

5'GAAGTTCCTATTCcGAAGTTCCTATTCtctagaaaGtATAGGAACTTC3'

This segment is dispensable for excision but essential for integration, including Recombinase-mediated cassette exchange.[4]

Because the recombination activity can be targeted to a selected organ, or a low level of recombination activity can be used to consistently alter the DNA of only a subset of cells, Flp-FRT can be used to construct genetic mosaics in multicellular organisms. Using this technology, the loss or alteration of a gene can be studied in a given target organ of interest, even in cases where experimental animals would not survive the loss of this gene in other organs("spatial control"). The effect of altering a gene can also be studied over time, by using an inducible promoter to trigger the recombination activity late in development ("temporal control") - this prevents the alteration from affecting overall development of an organ, and allows single cells lacking the gene to be compared to normal neighboring cells in the same environment.

See also

References

  1. ↑ Zhu XD, Sadowski PD (1995). "Cleavage-dependent Ligation by the FLP Recombinase". Journal of Biological Chemistry 270 (39): 23044–54. doi:10.1074/jbc.270.39.23044. PMID 7559444. 
  2. ↑ Schlake T, Bode J (1994). "Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci". Biochemistry 33 (43): 12746–12751. doi:10.1021/bi00209a003. PMID 7947678. 
  3. ↑ Turan, S.; Kuehle, J.; Schambach, A.; Baum, C.; Bode, J. (2010). "Multiplexing RMCE: Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology". J. Mol. Biol. 402 (1): 52–69. doi:10.1016/j.jmb.2010.07.015. PMID 20650281. 
  4. ↑ Turan, S., Bode, J. (2011). "Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications". FASEB J. 25: 4088–4107. doi:10.1096/fj.11-186940. PMID 21891781. 


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