Functional siRNA expression from transfected PCR products.
RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. In mammalian systems, the sequence-specific RNAi effect has been observed by expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. We describe here a facile PCR based strategy for rapid synthesis of siRNA expression units and their testing in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells resulting in functional expression of siRNAs. This approach should prove useful for identification of optimal siRNA-target combinations and for multiplexing siRNA expression in mammalian cells.
Castanotto
Daniela
D
Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Li
Haitang
H
Rossi
John J
JJ
D015152:Blotting, Northern; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D015694:Gene Products, rev / Q000378:metabolism*; D020868:Gene Silencing; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D012342:RNA, Small Nuclear; D014162:Transfection
D017931:DNA Primers; D015694:Gene Products, rev; D008164:Luminescent Proteins; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D049452:Green Fluorescent Proteins