Under our experimental conditions (48 hr of continuous Flp activity) all the cells of the P compartment acquire the ßGal label.
The successful application of recombinase technologies to porcine genetics requires the demonstration of Cre and/or Flp activity in porcine cells and the efficient delivery of RRS sites and recombinase-based expression vectors to the porcine genome.
In order to further evaluate the molecular mechanisms of FLP anticancer activities, we treated A549 lung carcinoma cells, with FLP or CTX treated rat derived serum (see cell culture methods).
The Cre(R292A) activity is distinct from the type II endonuclease activity of Flp(R308A) whose target is the scissile MeP bond in DNA rather than the cleaved MeP-tyrosyl intermediate [19].
The reaction is independent of the Flp active site nucleophile Tyr-343.
Here, the positively charged Arg-308 appears to deter water, as indicated by the hydrolytic activity of Flp(R308A) on MeP-DNA.
The activities of Flp and topoisomerase, as well as their mutant derivatives, on P and MeP containing DNA substrates are suggestive of a potential role for phosphate or active site electrostatics in blockading water nucleophile during phosphoryl transfer in these two systems [19] [21].
As a result, a new version of FLP, FLPe exhibited 4 10 fold higher activity than FLP at 37°C [6].
Initial use of FLP in mammalian cells revealed inefficient recombinase activity due to thermo-instability of the protein [6].
