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Use of Sindbis virus for genetic studies of insect development

Project:  Use of Sindbis virus for genetic studies of insect development
Research institute: Institute of Entomology,AS CR
With the powerful Drosophila model at hand, research on other insects might seem redundant. However, genetic studies on additional species are necessary for us to understand the general developmental mechanisms and how they evolved. One of our goals is to expand reverse genetic techniques so that causal evidence for a gene function may be obtained in any relevant invertebrate. Specifically, we are interested in genes that govern insect metamorphosis in response to the steroid hormone ecdysone. In Drosophila, ecdysone triggers metamorphosis by acting on its nuclear receptor, which in turn activates a cascade of transcription factors. Among these, Broad-Complex (BR-C) is specifically required for metamorphic events including pupation, extension of adult appendages, differentiation of eyes, and death of obsolete larval organs. Only loss-of-function studies in non-drosophilid species can answer the question whether the role of BR-C (or any other protein) is common to diverse insect orders. RNA interference (RNAi) is the most promising approach to loss-of-function genetic analyses in non-model organisms, where targeted mutagenesis is not feasible. The main problem is how to deliver the interfering double-stranded RNA, sufficient for a specific gene knock-down, into the organism cells. We have solved this problem by using a recombinant virus Sindbis, which infects some of insect tissues without killing the host. Once in the host cell, the Sindbis RNA genome (see Figure, top) including an inserted foreign gene is rapidly expressed, producing either protein (such as GFP) or double-stranded RNA that can trigger degradation of a specific endogenous mRNA and thereby silence the gene. We have demonstrated Sindbis-mediated RNAi in vivo using the silkworm Bombyx mori as a pilot system. BR-C RNAi disrupted silkworm pupation as well as the morphogenetic (differentiation of the adult wings, eyes and legs, see Figure) and degenerative (programed death of the larval silk glands) aspects of metamorphosis. These effects correspond to those of BR-C mutations in Drosophila, suggesting that the role of BR-C in metamorphosis is evolutionarily conserved (Uhlirova et al. 2003, PNAS USA). Our next goal is to use the Sindbis technology to address the function of ecdysone response genes across less advanced insect orders.


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