DNA substrates influence the recombination efficiency mediated by FLP recombinase expressed in mammalian cells

Abstract

The FLP recombinase derived from Saccharomyces cerevisiae mediates precise site-specific recombination between a pair of FLP recognition targets (FRTs). Like the Cre/loxP system derived from bacteriophage P1, the FLP/FRT system has recently been applied to gene regulation systems using an FLP-expressing recombinant adenovirus (rAd) (Nakano et al, Nucleic Acids Res. 29: e40, 2001). In an attempt to improve the FLP/FRT system by altering its DNA substrates, we compared the recombination efficiency among different substrates by a quantitative in vitro assay using FLP expressed in mammalian cells. Unexpectedly, we found that one linearized DNA substrate showed 4- to >20-fold lower recombination efficiency than other substrates, which phenomenon has not been observed in the Cre/loxP system. The quantitative in vitro assay using truncated DNA substrates suggested that the recombination efficiency seemed to be influenced not only by the linearized position of the substrate, but also by the length between a pair of FRTs. Such substrate preference of FLP expressed in mammalian cells should probably be noted when designing versatile applications of the FLP/FRT system as a gene regulation system in mammalian systems. Fortunately, however, we demonstrated that no substrate preference was observed when using a particular substrate (pCAFNF5) and the preference was reduced when using a certain pair of mutant FRTs (f72), which will also be a promising tool for simultaneous gene regulation in combination with wild-type FRT.