In this issue of TechNotes, Ambion introduces its pSilencer™ plasmid vector for the stable expression of siRNAs in mammalian cells. The use of such vectors has already been described in the literature by several groups (see the reference list below). Two of those papers are summarized here.

Paddison PJ, Caudy AA, and Hannon GJ (2002) Stable Suppression of Gene Expression by RNAi in Mammalian Cells. Proc. Natl. Acad. Sci. USA 99:1443­1448.

Brummelkamp TR, Bernards R, and Agami R (2002) A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science 296: 550­553.

RNA interference (RNAi), a sequence-specific post-transcriptional gene silencing phenomenon, occurs in an evolutionarily diverse group of organisms including plants, fungi, and metazoans. Cellular machinery involved in RNAi has been harnessed as an experimental tool to study gene function. Long dsRNA and 21-23-mer small interfering RNA have been utilized in non-mammalian and mammalian cells respectively to effect RNAi. The transient nature of the action of both types of dsRNA precludes the examination of phenotypes of dsRNA-induced gene silencing over long time periods. To overcome this limitation, several groups of investigators have designed plasmid vectors to direct the stable expression of dsRNA in mammalian cells. Hannon and colleagues first showed that 500 nt dsRNA induced gene-specific silencing responses in cultured murine cells. Further, they demonstrated that such dsRNA-induced gene silencing could be sustained continuously by stably transfecting these cells with plasmid vectors that direct the expression of long RNA hairpins from an RNA polymerase III promoter.

In contrast to the long RNA hairpins, Agami and colleagues constructed a vector to direct the expression of short RNA hairpins from the polymerase-III H1-RNA gene promoter. Both transient and stable transfections of cells with these expression vectors produced sense and antisense RNA strands, reduced both mRNA and protein levels of the target gene, and abrogate the cellular function of the target gene. These responses were highly sequence-specific, as a single nucleotide mismatch in the target sequence abolished the gene-silencing effects. In summary, vectors expressing long and short RNA hairpins should allow for stable and heritable gene silencing, thereby permitting the analysis of phenotypes that develop over long time periods. Since these reports, several other papers have appeared describing stable expression systems using plasmid vectors (see the list of references below).

RNArticles highlight articles published in leading peer-reviewed journals that are of particular interest to the RNA researchers.

1. Brummelkamp TR, Bernards R, and Agami R (2002) A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science 296: 550­553.

2. Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, Rossi J (2001) "Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells," Nature Biotechnology 19: 500­505.

3. Miyagishi M & Taira K (2002) "U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells," Nature Biotechnology 19: 497­500.

4. Paddison PJ, Caudy AA, and Hannon GJ (2002) Stable Suppression of Gene Expression by RNAi in Mammalian Cells. Proc. Natl. Acad. Sci. USA 99: 1443­1448.

5. Paul CP, Good PD, Winer I, Engelke DR (2002) "Effective Expression of Small Interfering RNA in human cells," Nature Biotechnology 19: 505­508.

6. Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC, and Shi Y. (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 99(8): 5515­5520.

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