Construction and transfection of PCR products expressing siRNAs or shRNAs in mammalian cells.
In mammalian cells, the RNA interference (RNAi) effect has been observed through expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. Here, we describe a facile polymerase chain reaction (PCR)-based strategy for rapid synthesis and evaluation of small interfering RNAs (siRNA) expression units in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells for functional testing. This method is fast and inexpensive, allowing several different siRNA gene candidates to be rapidly screened for efficacy.
Castanotto
Daniela
D
Departmetn of Biology and Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA, USA.
Rossi
John J
JJ
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D008322:Mammals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics*; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*
D017931:DNA Primers; D034741:RNA, Small Interfering