dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference.
RNA interference (RNAi) is a form of post-transcriptional gene silencing that has been described in a number of plant, nematode, protozoan, and invertebrate species. RNAi is characterized by a number of features: induction by double stranded RNA (dsRNA), a high degree of specificity, remarkable potency and spread across cell boundaries, and a sustained down-regulation of the target gene. Previous studies of RNAi have examined this effect in whole organisms or in extracts thereof; we have now examined the induction of RNAi in tissue culture. A screen of mammalian cells from three different species showed no evidence for the specific down-regulation of gene expression by dsRNA. By contrast, RNAi was observed in Drosophila Schneider 2 (S2) cells. Green fluorescent protein (GFP) expression in S2 cells was inhibited in a dose-dependent manner by transfection of dsRNA corresponding to gfp when GFP was expressed either transiently or stably. This effect was structure- and sequence-specific in that: (1) little or no effect was seen when antisense (or sense) RNA was transfected; (2) an unrelated dsRNA did not reduce GFP expression; and (3) dsRNA corresponding to gfp had no effect on the expression of an unrelated target transgene. This invertebrate tissue culture model should allow facile assays for loss of function in a well-defined cellular system and facilitate further understanding of the mechanism of RNAi and the genes involved in this process.
Caplen
N J
NJ
Clinical Gene Therapy Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.
Fleenor
J
J
Fire
A
A
Morgan
R A
RA
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D002478:Cells, Cultured; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004330:Drosophila / Q000166:cytology / Q000187:drug effects* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009843:Oligoribonucleotides / Q000494:pharmacology; D012330:RNA, Double-Stranded / Q000008:administration & dosage* / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004274:DNA, Recombinant; D008164:Luminescent Proteins; D009843:Oligoribonucleotides; D012330:RNA, Double-Stranded; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase