Large-scale production of dsRNA and siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase

Antti P Aalto, L Peter Sarin, Alberdina A van Dijk, Mart Saarma, Minna M Poranen, Urmas Arumäe, Dennis H Bamford

Research output: Contribution to journalArticleScientificpeer-review

54 Citations (Scopus)

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 6 to generate dsRNA molecules of practically unlimited length, and (2) an in vivo RNA replication system based on carrier state bacterial cells containing the 6 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.

Original languageEnglish
Pages (from-to)422-9
Number of pages8
JournalRNA
Volume13
Issue number3
DOIs
Publication statusPublished - Mar 2007
MoE publication typeA1 Journal article-refereed

Keywords

  • Animals
  • Bacteriophage phi 6/enzymology
  • Cell Line
  • Humans
  • Nucleic Acid Amplification Techniques/methods
  • RNA Interference
  • RNA Replicase/chemistry
  • RNA, Double-Stranded/biosynthesis
  • RNA, Small Interfering/biosynthesis
  • Viral Proteins/chemistry

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