A host of classical and molecular genetic tools make Drosophila a tremendous model for the dissection of gene activity. In particular, the FLP-FRT technique for mitotic recombination has greatly enhanced gene loss-of-function analysis. This technique efficiently induces formation of homozygous mutant clones in tissues of heterozygous organisms. However, the dependence of the FLP-FRT method on cell division, and other constraints, also impose limits on its effectiveness. We describe here the generation and testing of tools for Mutant Analysis by Rescue Gene Excision (MARGE), an approach whereby mutant cells are formed by loss of a rescue transgene in a homozygous mutant organism. Rescue-transgene loss can be induced in any tissue or cell-type and at any time during development or in the adult using available heat-shock-induced or tissue-specific flippases, or combinations of UAS-FLP with Gal4 and Gal80ts reagents. The simultaneous loss of a constitutive fluorescence marker (GFP or RFP) identifies the mutant cells. We demonstrate the efficacy of the MARGE technique by flip-out (clonal and disc-wide) of a Ubi-GFP-carrying construct in imaginal discs, and by inducing a known yki mutant phenotype in the Drosophila ovary.
Keywords: CKO; Flip-out Rescue Cassette vectors; FoRC vectors; MARGE; clonal analysis; conditional knockout; follicle cells; yorkie.
© 2016 Wiley Periodicals, Inc.