Effective expression of small interfering RNA in human cells.
In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a double-stranded RNA (dsRNA) corresponding to exon sequences in the mRNA (refs 1,2). The use of this "RNA interference" (RNAi) in mammalian studies had lagged well behind its utility in lower animals because uninterrupted RNA duplexes longer than 30 base pairs trigger generalized cellular responses through activation of dsRNA-dependent protein kinases. Recently it was demonstrated that RNAi can be made to work in cultured human cells by introducing shorter, synthetic duplex RNAs (approximately 20 base pairs) through liposome transfection. We have explored several strategies for expressing similar short interfering RNA (siRNA) duplexes within cells from recombinant DNA constructs, because this might allow long-term target-gene suppression in cells, and potentially in whole organisms. Effective suppression of target gene product levels is achieved by using a human U6 small nuclear RNA (snRNA) promoter to drive nuclear expression of a single RNA transcript. The siRNA-like parts of the transcript consists of a 19 base pair siRNA stem with the two strands joined by a tightly structured loop and a U1-4 3' overhang at the end of the antisense strand. The simplicity of the U6 expression cassette and its widespread transcription in human cell types suggest that this mode of siRNA delivery could be useful for suppressing expression of a wide range of genes.
Paul
Cynthia P
CP
Department of Biological Chemistry, The University of Michigan, Ann Arbor, MI 48109-0606, USA.
Good
Paul D
PD
Winer
Ira
I
Engelke
David R
DR
D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear / Q000378:metabolism*; D022661:RNA, Untranslated / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; D022661:RNA, Untranslated; D011994:Recombinant Proteins; C405486:U6 small nuclear RNA; D012313:RNA