MiMIC-TIFF: A Method for Making Gene-specific Gal4 lines from MiMIC Insertions into Coding Introns.

MiMIC-TIFF: A Method for Making Gene-specific Gal4 lines from MiMIC Insertions into Coding Introns. Fengqiu Diao¹, Feici Diao¹, Sarah Naylor¹, Holly Ironfield², Matthias Landgraf², Benjamin White¹. 1) Laboratory of Molecular Biology, NIMH, NIH, Bethesda, MD 20892; 2) University of Cambridge, Dept. of Zoology, Cambridge, UK.

The viral T2A sequence can promote translation of two proteins from a single mRNA, a feature that can be exploited to generate transgenic driver lines that express Gal4 in the same pattern as an endogenous gene of interest (Diao & White, 2012). This technique (called T2A-Gal4-In-Frame-Fusion, or T2A-GIFF) requires insertion of the T2A-Gal4 coding sequence into the reading frame of the endogenous gene by homologous recombination, or by recombineering suitable P[acman] clones and re-introducing them into the fly. But both of these methods are labor intensive. We show here that T2A-Gal4 (and other T2A-transgenes) can be readily introduced into genes of interest that contain a MiMIC insertion in a coding intron. Because more than 50% of intragenic MiMIC insertions lie within such introns (Venken KJT et al, 2010), this method, which we call MiMIC-T2A-mediated In-Frame-Fusion (i.e. MiMIC-TIFF), should be widely applicable. To implement MiMIC-TIFF, we have adapted protein-trap plasmids that allow the T2A-Gal4 sequence to be introduced in-frame into MiMIC insertions by C31- recombinase-mediated cassette exchange. To confirm the efficacy of the approach, we have generated transgenic lines by inserting T2A-Gal4 into multiple genes, including Rdl^MI02620, Shaw^MI01735 and GluRII B^MI03631 each of which requires a T2A-Gal4 fusion in a different reading frame. When crossed with a UAS-EYFP reporter, each of these lines shows strong expression in a pattern that matches that of the endogenous gene. Additional constructs, which allow creation of Gal80 and Split Gal4 hemidriver lines have also been made. To simplify the conversion of MiMIC lines into Gal4 driver lines, we are also working on dual-recombinase system using both cre and C31 that allows Gal4 drivers to be made in vivo, by a series of crosses. The utility of MiMIC-TIFF can be expected to increase together with the number of MiMIC insertions generated by the Drosophila Gene Disruption Project (Bellen et al., 2011).