Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage ϕ6 RNA-dependent RNA polymerase

  1. Antti P. Aalto1,2,3,
  2. L. Peter Sarin1,3,
  3. Alberdina A. van Dijk1,4,
  4. Mart Saarma2,
  5. Minna M. Poranen1,
  6. Urmas Arumäe2, and
  7. Dennis H. Bamford1
  1. 1Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Biocenter 2, FIN-00014 University of Helsinki, Helsinki, Finland
  2. 2Institute of Biotechnology, University of Helsinki, Biocenter 1, FIN-00014 University of Helsinki, Helsinki, Finland

  1. ↵3 These authors have contributed equally to this work.

Abstract

The discovery of RNA interference (RNAi) has revolutionized biological research and has a huge potential for therapy. Since small double-stranded RNAs (dsRNAs) are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. We present two novel, flexible virus-based systems for the efficient production of dsRNA: (1) an in vitro system utilizing the combination of T7 RNA polymerase and RNA-dependent RNA polymerase (RdRP) of bacteriophage ϕ6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the ϕ6 polymerase complex to produce virtually unlimited amounts of dsRNA of up to 4.0 kb. We show that pools of small interfering RNAs (siRNAs) derived from dsRNA produced by these systems significantly decreased the expression of a transgene (eGFP) in HeLa cells and blocked endogenous pro-apoptotic BAX expression and subsequent cell death in cultured sympathetic neurons.

Keywords

Footnotes

  • ↵4 Present address: School of Biochemistry, North-West University, Private Bag X6001, 2520 Potchefstroom, South Africa.

  • Reprint requests to: Dennis H. Bamford, Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, Biocenter 2, P.O. Box 56, FIN-00014 University of Helsinki, Helsinki, Finland; e-mail: dennis.bamford{at}helsinki.fi; fax: 358-9-19159098.

  • Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.348307.

    • Received October 13, 2006.
    • Accepted November 29, 2006.
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  1. Published in Advance January 19, 2007, doi: 10.1261/rna.348307 RNA 13: 422-429 Copyright © 2007 RNA Society

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