Flp recombinase mediated cassette exchange (RMCE) is an efficient method for inserting large DNA sequences at specific sites in the C. elegans genome. The method takes advantage of both the Flp/FRT system and the Cre/loxP system to integrate plasmid derived sequences into a specific locus called a “landing site”. The source of Flp can either be provided by a locus unlinked to the integration landing site (A), or incorporated into the landing site (B).
In brief, the method consists of six steps (above) each of which is monitored by visual screens. First, the insert of interest is cloned into a specialized vector that contains loxP, FRT and FRT3 sites as well as a self-excising cassette (SEC) that also expresses the sqt-1(e1350) visible marker. Second, the plasmid is injected into a strain expressing FLP in the germline, and harboring a loxP, FRT and FRT3 tagged landing site that expresses GFP-his-58 under the control of the ubiquitous rpl-28 promoter. Third, Rol progeny of the injected animals are identified and pooled. Fourth, the progeny of the pooled animals are screened for Rol F2 progeny. Greater than 90% of F2 Rol progeny are integration events. Fifth, homozygous integrants are identified as somatic GFP(-) F3 progeny. Sixth, the SEC is excised by heat shock yielding the final integrant. In my hands, the rate of insertion is greater than 1 per 3 injected P0 animals. Two versions of the protocol have been established. If one uses an unlinked source of Flp that must be crossed out after isolating the integrant. The procedure takes about two weeks to perform once you have the appropriate integration clone in hand (see timeline below) and requires a 1/2 to 1 hr of hands on labor per generation to perform.
