Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.
RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.
Lee
Nan Sook
NS
Division of Molecular Biology, Graduate School of Biological Sciences, City of Hope, Duarte, CA 91010, USA.
Dohjima
Taikoh
T
Bauer
Gerhard
G
Li
Haitang
H
Li
Ming-Jie
MJ
Ehsani
Ali
A
Salvaterra
Paul
P
Rossi
John
J
D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D015694:Gene Products, rev / Q000378:metabolism*; D005821:Genetic Techniques; D006801:Humans; D050356:Lipid Metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012330:RNA, Double-Stranded; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering; D022661:RNA, Untranslated / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D015694:Gene Products, rev; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D022661:RNA, Untranslated