Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

  1. Patrick J. Paddison1,
  2. Amy A. Caudy1,
  3. Emily Bernstein2,3,
  4. Gregory J. Hannon1,2,4, and
  5. Douglas S. Conklin2
  1. 1Watson School of Biological Sciences, 2Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA; 3Graduate Program in Genetics, State University of New York at Stony Brook, Stony Brook, New York 11794, USA

Abstract

RNA interference (RNAi) was first recognized inCaenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (∼70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.

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Footnotes

  • 4 Corresponding author.

  • E-MAIL hannon{at}cshl.org; FAX (516) 367-8874.

  • Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.981002.

    • Received January 31, 2002.
    • Accepted March 8, 2002.
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