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U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells

Nature Biotechnology volume 20, pages 497–500 (2002)Cite this article

Abstract

The first evidence for gene disruption by double-stranded RNA (dsRNA) came from careful analysis in Caenorhabditis elegans1. This phenomenon, called RNA interference (RNAi), was observed subsequently in various organisms, including plants, nematodes, Drosophila, and protozoans2,3,4,5. Very recently, it has been reported that in mammalian cells, 21- or 22-nucleotide (nt) RNAs with 2-nt 3′ overhangs (small inhibitory RNAs, siRNAs) exhibit an RNAi effect6,7. This is because siRNAs are not recognized by the well-characterized host defense system against viral infections, involving dsRNA-dependent inhibition of protein synthesis. However, the current method for introducing synthetic siRNA into cells by lipofection restricts the range of applications of RNAi as a result of the low transfection efficiencies in some cell types and/or short-term persistence of silencing effects8. Here, we report a vector-based siRNA expression system that can induce RNAi in mammalian cells. This technical advance for silencing gene expression not only facilitates a wide range of functional analysis of mammalian genes but might also allow therapeutic applications by means of vector-mediated RNAi.

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Figure 1: Strategy for generating siRNA using U6 promoter.
Figure 2: Effect of siRNA expression vector targeted against various reporter genes.
Figure 3: Analysis of a series of siRNA expression vectors targeted against reporter genes or an endogenous gene.

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Acknowledgements

This research was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, and grants from the Ministry of Economy, Trade and Industry (METI) of Japan.

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Authors and Affiliations

  1. Department of Chemistry and Biotechnology, School of Engineering, the University of Tokyo, Hongo, 113-8656, Tokyo, Japan

    Makoto Miyagishi & Kazunari Taira

  2. Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba Science City, 305-8562, Japan

    Kazunari Taira

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  1. Makoto Miyagishi
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Correspondence to Kazunari Taira.

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Miyagishi, M., Taira, K. U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 20, 497–500 (2002). https://doi.org/10.1038/nbt0502-497

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