A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila

Abstract

Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

“… Representative images (left) and quantifications (right) of the number of intestinal progenitor cells in control midguts or midguts in which Ret has been downregulated from adult ISCs/EBs [achieved by esg‐Gal4 , tub‐Gal80 TS ‐driven Ret‐RNAi, enhanced by UAS‐Dicer2 ( Dcr2 ) co‐expression (Dietzl et al , 2007)] for 4, 10 or 20 days.MARCM clone size quantifications (graph) and representative images (clones labelled in green with GFP) reveal that clones lacking Ret ( Ret KO ) or expressing Ret‐RNAi are smaller than control clones 10 days after clone induction.Quantifications of mitoses (pH3‐positive cells, graph) and visualisation of intestinal progenitors (using esg ‐driven GFP, image panels) in midguts of flies with the same genotypes as in (A). The regenerative response triggered by damage‐inducing DSS in control flies is reduced following Ret downregulation from ISC/EBs.…”

“…Representative images (left) and quantifications (right) of the number of intestinal progenitor cells in control midguts or midguts in which Ret has been downregulated from adult ISCs/EBs [achieved by esg‐Gal4 , tub‐Gal80 TS ‐driven Ret‐RNAi, enhanced by UAS‐Dicer2 ( Dcr2 ) co‐expression (Dietzl et al , 2007)] for 4, 10 or 20 days.…”

“…UAS‐Flybow 1.1 [Bloomington Drosophila Stock Centre (BDSC): 35537, (Hadjieconomou et al , 2011)], UAS‐StGFP ( UAS‐Stinger , (Barolo et al , 2000), gift from M. Landgraf), UAS‐Dcr2 ( UAS‐Dicer2 , (Dietzl et al , 2007), gift from I. Salecker), UAS‐Ret [ UAS‐Ret‐3xFLAG‐6xHis (Kallijarvi et al , 2012)]; UAS‐upd1 (Jiang et al , 2009; gift from J. Cordero); UAS‐sspitz [Schweitzer et al , 1995; gift from J. Treisman); UAS‐wg ( UAS‐wg::HA , BDSC:5918); UAS‐sgg DN ( UAS‐sgg.A81T , BDSC: 5360, (Bourouis, 2002)]; UAS‐dsh DN ( UAS‐dshΔB21‐2 , (Axelrod et al , 1998), gift from J. Axelrod); UAS‐pan DN (van de Wetering et al , 1997; gift from F.J. Díaz‐Benjumea); UAS‐Src42A CA (BDSC: 6410); UAS‐Dcr2 (VDRC Stock Centre: 60007); UAS‐Ret‐RNAi (VDRC: GD 843); UAS‐dsh‐RNAi (VDRC: KK 101525); UAS‐Src42A‐RNAi (VDRC:KK 100708); UAS‐Src64B‐RNAi (VDRC: GD 35252), UAS‐Fak (Palmer et al , 1999). …”

A male‐specific effect of dominant‐negative Fos

“…When vg-Fbz was crossed to mutations in mle, msl-3, and the triple recombinant chromosome containing alleles of msl-2, msl-1, and mle we observed a significant increase in the percentage of defective male wings (Table 1): mle (71%), msl-3 (71.5%), and msl-2 msl-1 mle (73%). We also tested a UAS-regulated RNAi transgene directed against msl-3 RNA (Dietzl et al, 2007). When this construct was driven by means of Vg-Gal4, a low level (4.5%) of wing defects was evident, limited to males.…”

“…roX1 ex21A wϩ4⌬4.3/wϩ4⌬4.3, and the recombinant hs-Msl-1-Z1ϩhs-Msl-2-6I were obtained from V. Meller (Wayne State). A UAS-regulated RNAi transgene directed against msl-3 RNA was obtained from the VDRC (Dietzl et al, 2007). For genetic interaction experiments UASFbz was recombined onto chromosome 2 with the vg-Gal4 driver, creating the vg-Fbz chromosome.…”