Question

What primer can be used to assess a gene that has been knocked down using UAS-RNAi in CNS(Elav-Gal4) of Drosophila?

Answer 1

Targeted RNA interference, or RNAi, is a widely used experimental technique for gene silencing that has been successfully applied to identify novel genes that control stem cell fate and proliferation in Drosophila. While the creation of genetic mutant alleles can be time-consuming and labor intensive, generation of RNAi against a target of interest is relatively easy and inexpensive. Further, while complete loss-of-function genetic mutant alleles are useful tools, they may result in severe phenotypes that are difficult to interpret or developmentally lethal, precluding analysis of gene function in adult cells. In contrast, RNAi frequently reduces, but does not abolish, the targeted mRNA, resulting in hypomorphic phenotypes. By inducing RNAi using the UAS-GAL4system, spatial control over gene knockdown in developing or adult fly tissues is readily accomplished . In this system, promoter or enhancer elements drive the expression of the yeast transcription factor Gal4 . Once expressed, Gal4 binds a DNA response element, the upstream activating sequence(UAS), which precedes a gene fragment containing an inverted repeat with exact complementarity to a given gene of interest. Expression of the inverted repeat results in the formation of double-stranded RNA hairpins that trigger a sequence-specific RNAi silencing response in the cells in which it is expressed. Thus, RNA hairpins are generated under the control of tissue- or cell-specific drivers, allowing for targeted knockdown of a gene of interest in specific cell population(s). RNAi experiments can be combined with additional genetic tools to restrict knockdown to specific temporal windows or to a clonal population of cells within a wild-type environment (mosaic analysis with a repressible cell marker; MARCM) . RNAi in Drosophila ovarian GSCs thus presents an elegant experimental system to discover the molecular regulations that govern stem cell fate and function in vivo.