Gene knock-down using RNAi delivered by a virus

RNA interference (RNAi) allows gene silencing, and thus analyses of gene function, even 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 degradation of the target mRNA, into the organism's cells. We have solved this problem using a recombinant virus Sindbis. Once in host cells, the Sindbis RNA genome including an inserted foreign gene is replicated in both directions, such that resulting double-stranded RNA can trigger degradation of the endogenous mRNA for this gene and thereby silence it. We have for the first time demonstrated this process in vivo in the commercial silkworm. By silencing a gene for the transcription factor Broad-Complex (BR-C), we disrupted pupation of the silkworm (figure) and two classes of processes during metamorphosis: (i) differentiation of the adult wings, eyes and legs from their larval primordia, and (ii) programed death of the larval silk glands. These defects correspond to the effects of BR-C mutations in Drosophila and thus show that the role of BR-C in insect metamorphosis is evolutionarily conserved. In addition, our results have demonstrated the usefulness of the Sindbis virus as a vehicle for genetic "knock-outs" in non-model species. (Uhlirova et al. 2003; P.N.A.S. 100, 15607-15612).