Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

Effective expression of small interfering RNA in human cells

Nature Biotechnology volume 20, pages 505–508 (2002)Cite this article

Abstract

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 kinases3. Recently it was demonstrated4 that RNAi can be made to work in cultured human cells by introducing shorter, synthetic duplex RNAs (∼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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expression cassettes and small RNA inserts.
Figure 2: Effects of siRNA constructs on lamin A/C levels.
Figure 3: Localization of U6+27 siRNA transcripts.

Similar content being viewed by others

References

  1. Zamore, P.D. RNA interference: listening to the sound of silence. Nat. Struct. Biol. 8, 746–750 (2001).

    Article  CAS  Google Scholar 

  2. Bernstein, E., Denli, A.M. & Hannon, G.J. The rest is silence. RNA 7, 1509–1521 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Gil, J. & Esteban, M. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis 5, 107–114 (2000).

    Article  CAS  Google Scholar 

  4. Elbashir, S.M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  5. Good, P.D. et al. Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther. 4, 45–54 (1997).

    Article  CAS  Google Scholar 

  6. Bertrand, E. et al. The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA 3, 75–88 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Cheong, C., Varani, G. & Tinoco, I. Jr. Solution structure of an unusually stable RNA hairpin, 5′-GGAC(UUCG)GUCC. Nature 346, 680–682 (1990).

    Article  CAS  Google Scholar 

  8. Elbashir, S.M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

    Article  CAS  Google Scholar 

  9. Nykänen, A., Haley, B. & Zamore, P.D. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309–321 (2001).

    Article  Google Scholar 

  10. Lipardi, C., Wei, Q. & Paterson, B.M. RNAi as random degradative PCR: siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs. Cell 107, 297–307 (2001).

    Article  CAS  Google Scholar 

  11. Chatterjee, S., Johnson, P.R. & Wong, K.K. Jr. Dual-target inhibition of HIV-1 in vitro by means of an adeno-associated virus antisense vector. Science 258, 1485–1488 (1992).

    Article  CAS  Google Scholar 

  12. Kunkel, G.R., Maser, R.L., Calvet, J.P. & Pederson, T. U6 small nuclear RNA is transcribed by RNA polymerase III. Proc. Natl. Acad. Sci. USA 83, 8575–8579 (1986).

    Article  CAS  Google Scholar 

  13. Kunkel, G.R. & Pederson, T. Upstream elements required for efficient transcription of a human U6 RNA gene resemble those of U1 and U2 genes even though a different polymerase is used. Genes Dev. 2, 196–204 (1988).

    Article  CAS  Google Scholar 

  14. Danzeiser, D.A., Urso, O. & Kunkel, G.R. Functional characterization of elements in a human U6 small nuclear RNA gene distal control region. Mol. Cell. Biol. 13, 4670–4678 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by NIH grant AI40936 to D.R.E. and the Medical Scientist Training Program at the University of Michigan. We thank Gary R. Kunkel for the cloned, human U6 snRNA gene, and Michael Imperiale and Eric Fearon for other materials.

Author information

Authors and Affiliations

  1. Department of Biological Chemistry, The University of Michigan, Ann Arbor, 48109-0606, MI

    Cynthia P. Paul, Paul D. Good & David R. Engelke

  2. Medical Scientist Training Program, The University of Michigan, Ann Arbor, 48109-0606, MI

    Ira Winer & David R. Engelke

Authors
  1. Cynthia P. Paul
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul D. Good
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ira Winer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David R. Engelke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David R. Engelke.

Ethics declarations

Competing interests

D.R.E., C.P.P., and P.D.G have filed a patent application for expression of si-like RNAs in mammalian cells using U6 promoter cassettes, entitled “Intracellular Expression and Delivery of siRNAs in Mammalian Cells.”

Rights and permissions

Reprints and permissions

About this article

Cite this article

Paul, C., Good, P., Winer, I. et al. Effective expression of small interfering RNA in human cells. Nat Biotechnol 20, 505–508 (2002). https://doi.org/10.1038/nbt0502-505

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0502-505

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing