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

Eric Billy, Vincent Brondani, Haidi Zhang, Ulrich Müller, Witold Filipowicz

Research output: Contribution to journalArticlepeer-review

340 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)14428-14433
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume98
Issue number25
DOIs
StatePublished - Dec 4 2001
Externally publishedYes

ASJC Scopus subject areas

  • General

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