TY - JOUR
T1 - Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines
AU - Billy, Eric
AU - Brondani, Vincent
AU - Zhang, Haidi
AU - Müller, Ulrich
AU - Filipowicz, Witold
PY - 2001/12/4
Y1 - 2001/12/4
N2 - In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression, referred to as RNA interference (RNAi). In invertebrates, RNAi can be triggered effectively by either long dsRNAs or 21- to 23-nt-long short interfering (si) duplex RNAs, acting as effectors of RNAi.siRNAs recently have been shown to act as potent inducers of RNAi in cultured mammalian cells. However, studies of RNAi activated by long dsRNA are impeded by its nonspecific effects, mediated by dsRNA-dependent protein kinase PKR and RNase L. Here, we report that the RNAi response can be induced effectively by long dsRNA in nondifferentiated mouse cells grown in culture. Transfection of dsRNA into embryonal carcinoma (EC) P19 and F9 cells results in a sequence-specific decrease in the level of proteins expressed from either exogenous or endogenous genes. dsRNA-mediated inhibition of the reporter gene also occurs in mouse embryonic stem cells. The RNAi effect is mediated by siRNAs, which are generated by cleavage of dsRNA by the RNaselll-like enzyme, Dicer. We demonstrate that extracts prepared from EC cells catalyze processing of dsRNA into ≈23-nt fragments and that Dicer localizes to the cytoplasm of EC and HeLa cells.
AB - In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression, referred to as RNA interference (RNAi). In invertebrates, RNAi can be triggered effectively by either long dsRNAs or 21- to 23-nt-long short interfering (si) duplex RNAs, acting as effectors of RNAi.siRNAs recently have been shown to act as potent inducers of RNAi in cultured mammalian cells. However, studies of RNAi activated by long dsRNA are impeded by its nonspecific effects, mediated by dsRNA-dependent protein kinase PKR and RNase L. Here, we report that the RNAi response can be induced effectively by long dsRNA in nondifferentiated mouse cells grown in culture. Transfection of dsRNA into embryonal carcinoma (EC) P19 and F9 cells results in a sequence-specific decrease in the level of proteins expressed from either exogenous or endogenous genes. dsRNA-mediated inhibition of the reporter gene also occurs in mouse embryonic stem cells. The RNAi effect is mediated by siRNAs, which are generated by cleavage of dsRNA by the RNaselll-like enzyme, Dicer. We demonstrate that extracts prepared from EC cells catalyze processing of dsRNA into ≈23-nt fragments and that Dicer localizes to the cytoplasm of EC and HeLa cells.
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U2 - 10.1073/pnas.261562698
DO - 10.1073/pnas.261562698
M3 - Article
C2 - 11724966
AN - SCOPUS:0035807818
SN - 0027-8424
VL - 98
SP - 14428
EP - 14433
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 25
ER -