Small RNA-mediated gene silencing controls a wide range of processes during development. So far, three classes of small RNAs have been characterized in Drosophila, including small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-interacting RNAs 3. Double-stranded siRNA-mediated gene silencing, as a potent tool, has been widely used for knocking down gene expression in S2 cell cultures and in multiple tissues in flies. In Drosophila oogenesis, the siRNA pathway is apparently activated during egg activation, because the introduction of exogenous dsRNAs results in degradation of target mRNAs. However, in the early stage of germ cell development these cells fail to support dsRNA-mediated RNA interference because of unknown inhibitory mechanisms. To our knowledge no practical protocol for siRNA-mediated gene knockdown has yet been established for the Drosophila ovarian germline.

The miRNAs are structurally similar to siRNAs, but have a distinct biogenesis mechanism and execute gene silencing either through degradation of target mRNAs or by translation repression of target genes. There is increasing evidence that the miRNA pathway plays critical roles in the regulation of GSCs and germ cell development, suggesting that miRNA-mediated gene silencing functions as an important mechanism in regulating Drosophila germline development 4, 5. Haley et al. 6 have now successfully established a simplified gene silencing method by using artificial miRNAs in Drosophila S2 cell cultures and in wing imaginal discs. Therefore, we sought to test whether artificial miRNAs could also be applied in germ cells for knocking down genes of interest and for systematic reverse genetic screening to identify hitherto undiscovered genes involved in germline development.

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