Modular and Excisable Molecular Switch for the Induction of Gene Expression by the Yeast FLP Recombinase

Abstract

Site-specific recombinases (SSRs) and their cognate target sequences have become instrumental in biological research (1,2). By varying the number, type, and orientation of SSR recognition sites, one can induce predictable genomic manipulations, like inversions, deletions, and insertions, in cells of virtually any organism. Prominent applications include: (i) conditional gene activation or inactivation to study gene function in cells and in whole organisms; (ii) generation of cell lines that encode cytotoxic products or synthesize constant amounts of proteins after DNA integration into predefined loci; and (iii) selection marker removal. The FLP/FRT system of Saccharomyces cerevisiae consists of an enzyme (FLP) that mediates highfidelity recombination between two 48-bp-long recognition sequences (FRT sites) (1,3). Plasmids containing one FRT motif can be inserted into a separate template containing another FRT site. In addition, FLP catalyzes the excision and inversion of DNA flanked by FRT sequences in a direct or inverted repeat configuration, respectively. In the former case, the excision product consists of a circularized molecule. Normally, recombinaseinducible gene expression relies on constructs containing upstream of the open reading frame (ORF), a promoter linked to a direct repeat of FRT sites framing a transcriptional terminator. Recombinase-mediated excision of the latter element leads to the synthesis of the desired product. Here, we generated and tested constructs harboring a FLPresponsive molecular switch with a different arrangement (i.e., with the promoter located downstream of the ORF) (Figure 1A). In this configuModular and excisable molecular switch for the induction of gene expression by the yeast FLP recombinase