%0 Journal Article
%T Short-term cytotoxic effects and long-term instability of RNAi delivered using lentiviral vectors
%A Richard J Fish
%A Egbert KO Kruithof
%J BMC Molecular Biology
%D 2004
%I BioMed Central
%R 10.1186/1471-2199-5-9
%X As expected, we encountered a cytotoxic phenotype when targeting the PAI-2 mRNA with vector-derived shRNA. However, this predicted phenotype was a potent non-specific effect of shRNA expression, as functional overexpression of the target protein failed to rescue the phenotype. By decreasing the shRNA length or modifying its sequence we maintained PAI-2 silencing and reduced, but did not eliminate, cytotoxicity. ShRNA of 21 complementary nucleotides (21 mers) or more increased expression of the oligoadenylate synthase-1 (OAS1) interferon-responsive gene. 19 mer shRNA had no effect on OAS1 expression but long-term selective pressure on cell growth was observed. By lowering lentiviral vector titre we were able to reduce both expression of shRNA and induction of OAS1, without a major impact on the efficacy of gene silencing.Our data demonstrate a rapid cytotoxic effect of shRNAs expressed in human tumor cell lines. There appears to be a cut-off of 21 complementary nucleotides below which there is no interferon response while target gene silencing is maintained. Cytotoxicity or OAS1 induction could be reduced by changing shRNA sequence or vector titre, but stable gene silencing could not be maintained in extended cell culture despite persistent marker gene expression from the RNAi-inducing transgene cassette. These results underscore the necessity of careful controls for immediate and long-term RNAi use in mammalian cell systems.Gene silencing is a powerful tool with which to study protein function. Gene inactivations in mice have revolutionised the way we study both basic biology and a plethora of disease types [1,2]. Gene silencing in human cells has, until recently, proven difficult to achieve [3]. Research with plants, flies and worms recently uncovered a mechanism by which eukaryotic cells target mRNAs, and perhaps even genetic loci, for specific gene silencing. This process is termed RNA interference (RNAi). RNAi can also be induced in mammalian cells using double-
%U http://www.biomedcentral.com/1471-2199/5/9