Flip-Flop

A cell cycle-independent, conditional gene inactivation strategy for differentially tagging wild-type and mutant cells.  eLife 6: e26420. 
Here, we describe a novel method based on intronic MiMIC insertions described in Nagarkar-Jaiswal et al. (2015) to perform conditional gene inactivation in Drosophila.  Mosaic analysis in Drosophila cannot be easily performed in post-mitotic cells.  We, therefore, developed Flip-Flop, a flippase-dependent in vivo cassette-inversion method that marks wild-type cells with the endogenous EGFP-tagged protein, whereas mutant cells are marked with mCherry upon inversion.  We document the ease and usefulness of this strategy in differential tagging of wild-type and mutant cells in mosaics.  We use this approach to phenotypically characterize the loss of SNF4Aγ, encoding the γ subunit of the AMP Kinase complex.  The Flip-Flop method is efficient and reliable, and permits conditional gene inactivation based on both spatial and temporal cues, in a cell cycle-, and developmental stage-independent fashion, creating a platform for systematic screens of gene function in developing and adult flies with unprecedented detail. 

フリップフロップ、ていうとビーチサンダルなんだけど、これは新しいflippase-inducedのmosaic system。このFlip-Flop cassetteの構造は以下の通り。
This cassette contains two modules that are placed in opposite orientation: a protein-trap (PT) module and a gene-trap (GT) module.  The PT module carries a splice acceptor (SA), followed by an in-frame EGFP coding sequence, and a splice donor (SD).  The GT module similarly contains a SA, but is followed by the T2A peptide sequence, an mCherry coding sequence, stop codons in all three reading frames, and an SV40 polyA signal.  The PT and GT modules are placed in opposite orientations and are flanked by inverted pairs of canonical FRT and FRT14 sites, forming a flip-excision switch (FLEx). 

両端にattBが付いているので、Recombination-Mediated Cassette Exchangeを利用して目的の遺伝子のイントロンに乗っているMiMIC (Minos-Mediated Integration Cassette)と載せ換える。これがprotein-trap orientationで入れば、目的の遺伝子のタンパクがEGFPでタグ付けされる。で、これにhsFLPでcassette inversionを誘導すると、EGFPは逆向きになってしまうので発現が消え、その代わりにそれまで逆向きに載っていたT2A-mCherryが発現することになり、transcriptionはその後ろについているstop codon & polyA sequenceで終わる。truncated transcriptはT2A siteがtranslational skipを誘導するので、mCherryだけがtranslateされるという仕組み。つまり、wild-type cloneはEGFPで、mutant cloneはmCherryでマークされる。
まぁ色々と想像はふくらむんやけど、やっぱりmitotic recombinationを利用するFLP-FRTと違って、post-mitotic cellsでもワークするというとこかね。