Double-Reciprocal Crossover Mediated by FLP-Recombinase: A Concept and an Assay,

Abstract

FLP recombinase induces a double-reciprocal crossover event between sets of different FLP recognition target (FRT) sites. Therefore, if these sites flank an expression cassette at a given genomic locus, it can be exchanged for another cassette that has been constructed in the analogous way [Schlake & Bode (1994) Biochemistry33, 12746−12751]. Here we demonstrate that an integrated expression cassette, flanked by a wild type and a mutated site, remains completely stable in the presence of constitutive FLP activity, obviating the need for a timing of this parameter. Therefore the only variable left for optimization is the initial concentration of the exchange plasmid. Since the exchange plasmid lacks a promoter, random integration is not expected to confer resistance to the selection marker, the expression of which requires the acquisition of the SV40 promoter provided at the predetermined integration site. Due to the presence of a luciferase reporter in a specific bicistronic expression cassette, recombination generates bioluminescence upon recombination, indicating the extent of the exchange reaction. This principle is utilized to compare the potential of various cell lines to support the exchange reaction and to adjust the optimum parameters.