Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibitio n of gene expression, referred to as RNA interference (RNAi). In invertebra tes, RNAi can be triggered effectively by either long dsRNAs or 21- to 23-n t-long short interfering (si) duplex RNAs, acting as effectors of RNAi. siR NAs recently have been shown to act as potent inducers of RNAi in cultured mammalian cells. However, studies of RNAi activated by long dsRNA are imped ed by its nonspecific effects, mediated by dsRNA-dependent protein kinase P KR and RNase L. Here, we report that the RNAi response can be induced effec tively by long dsRNA in nondifferentiated mouse cells grown in culture. Tra nsfection of dsRNA into embryonal carcinoma (EC) P19 and F9 cells results i n a sequence-specific decrease in the level of proteins expressed from eith er exogenous or endogenous genes. dsRNA-mediated inhibition of the reporter gene also occurs in mouse embryonic stem cells. The RNAi effect is mediate d by siRNAs, which are generated by cleavage of dsRNA by the RNaseIII-like enzyme, Dicer. We demonstrate that extracts prepared from EC cells catalyze processing of dsRNA into approximate to 23-nt fragments and that Dicer loc alizes to the cytoplasm of EC and HeLa cells.