RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase.
Methods that allow the specific silencing of a desired gene are invaluable tools for research. One of these is based on RNA interference (RNAi), a process by which double-stranded RNA (dsRNA) specifically suppresses the expression of a target mRNA. Recently, it has been reported that RNAi also works in mammalian cells if small interfering RNAs (siRNAs) are used to avoid activation of the interferon system by long dsRNA. Thus, RNAi could become a major tool for reverse genetics in mammalian systems. However, the high cost and the limited availability of the short synthetic RNAs and the lack of certainty that a designed siRNA will work present major drawbacks of the siRNA technology. Here we present an alternative method to obtain cheap and large amounts of siRNAs using T7 RNA polymerase. With multiple transfection procedures, including calcium phosphate co-precipitation, we demonstrate silencing of both exogenous and endogenous genes.
Donzé
Olivier
O
Département de Biologie Cellulaire, Université de Genève, Sciences III, 30 quai Ernest-Ansermet, CH-1211 Genève 4, Switzerland.
Picard
Didier
D
D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins
D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases