RNAi in the Hedgehog Signaling Pathway: pFRiPE, a Vector for Temporally and Spatially Controlled RNA

RNA interference (RNAi) has become an irreplaceable tool for reverse genetics in plants and animals. The universality and specificity of this phenomenon allows silencing of virtually any chosen gene to examine its involvement in biological processes. Many strategies exist to reduce the expression of a particular gene using RNAi. Some rely on delivering directly to cells the ∼21-nucleotide long interfering double-stranded RNA (dsRNA) species that are central mediators of the silencing process. Others rely on the transgenic expression of longer dsRNA molecules, leaving it to the cellular machinery to process these hairpins into short active dsRNA. In this chapter, we describe a transgenic method to deplete a chosen protein from a specific Drosophila tissue following induction of long dsRNA. It was used to uncover the role of lipidic particles in Hedgehog signaling by silencing lipophorin in the fat body (1 ), and we routinely use it to deplete specific proteins from wing imaginal disc subdomains (2 ). The method, certainly not restricted to the study of Hedgehog signaling, allows fast and efficient construction of a plasmid incorporating various Drosophila genetic tools to allow heat-shock-induced expression of dsRNA at the desired time and in the desired tissue. For protocols involving injection of in vitro synthesized dsRNA in embryos to study Hedgehog signaling, see for example (3 ). For genomic screens to identify Hedgehog pathway components in tissue culture cells by transfection of small interfering RNAs, see refs. (4 ,5 ).