Heritable gene silencing in Drosophila using double-stranded RNA

Abstract

RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants, nematodes, Drosophila, zebrafish, and mice. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene. Rapid degradation of mRNA in affected cells blocks gene expression. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.

JNK pathway mediates apoptotic cell death induced by tumor suppressor LKB1 in Drosophila

“…When we reduced LKB1 expression in the thorax region by a complementary RNA hairpin corresponding to LKB1 mRNA (LKBi), thoracic morphogenesis was significantly disrupted with bristle and scutellum formation defects ( Supplementary Figure 4a-d). 53 The same phenotypes were consistently observed with comparable penetrances (Supplementary Figure 4e) when dsRNAi was generated by other approaches such as usage of the hairpin construct with the inversed orientation (Inv) and of the SympUAST vector inducing symmetrical transcription to generate dsRNAi (Sym). 53,54 Therefore, we concluded that LKB1 is involved in the thoracic morphogenetic processes.…”

“…53 The same phenotypes were consistently observed with comparable penetrances (Supplementary Figure 4e) when dsRNAi was generated by other approaches such as usage of the hairpin construct with the inversed orientation (Inv) and of the SympUAST vector inducing symmetrical transcription to generate dsRNAi (Sym). 53,54 Therefore, we concluded that LKB1 is involved in the thoracic morphogenetic processes.…”

“…dsRNAi transgenes were constructed against 519-1104 base pairs of LKB1 coding sequence, as previously described. 53,54 For direct RNAi microinjection, dsRNAi against 971-1479 base pairs of LKB1 was synthesized and microinjected into embryos as described. 53 The Gal4 lines used in this study are Ay-Gal4, 58 ap-Gal4, 38 gmr-Gal4, 41 ms1096-Gal4, 41 hs-Gal4, 50 and pnr-Gal4.…”

“…53,54 For direct RNAi microinjection, dsRNAi against 971-1479 base pairs of LKB1 was synthesized and microinjected into embryos as described. 53 The Gal4 lines used in this study are Ay-Gal4, 58 ap-Gal4, 38 gmr-Gal4, 41 ms1096-Gal4, 41 hs-Gal4, 50 and pnr-Gal4. 30 Other stocks used in this study, UASHep, UAS-Bsk WT , and UAS-Bsk DN were gifts from Dr Mlodzik; puc E69 , which is also called puc-LacZ, was a gift from Dr Adachi-Yamada; UAS-DIAP was a gift from Dr Hay; UAS-Puc was a gift from Dr Peifer; LKB1 4A4À2 was a gift from Dr St Johnston; dpp-LacZ was a gift from Dr Kim; UAS-Eiger was obtained from Genexel Inc.; and bsk 1 , bsk 2 , hep r75 Df(3L)H99, UAS-Reaper, gmr-Hid, Ay4GFP (Ay-Gal4 UAS-GFP), UAS-GFP, and UAS-Msn were obtained from the Bloomington Drosophila Stock Center.…”

Transgene‐mediated RNA interference defines a novel role for notch in chemosensory startle behavior

“…However, several reports have recently demonstrated that the transcription of an inverted repeat from a transgene can form a hairpin loop in vivo. Furthermore, by utilizing the power of the Drosophila genetics and the GAL4/UAS binary system (Brand and Perrimon, 1993), one can express these hairpin RNAs in a temporal and spatialspecific manner in transgenic animals (Fortier and Belote, 2000;Kennerdell and Carthew, 2000).…”