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• W1999601787 abstract "RNA interference is widely used to silence gene expression in mammalian cells. We recently reported that an shRNA expressed from the H1 promoter in a lentiviral vector could induce the expression of a large group of interferon-stimulated genes (ISGs). This response was unrelated to silencing of the gene targeted by the shRNA MORF4L1. In parallel, we constructed lentiviral vectors expressing shRNA from the U6 promoter and found that these too could induce expression of OAS1, a classic interferon target gene. The U6 vectors give a higher frequency of ISG induction than comparable lentiviral H1 vectors, suggesting that there might be a fundamental flaw in the vector design. We have characterized the U6 vectors in detail and report here that ISG induction is a consequence of the presence of an AA di-nucleotide near the transcription start site. A single nucleotide deletion in the siRNA sequence abolished OAS1 induction, suggesting that the mechanism underlying the response uses a sensor that can detect 19 bp RNA duplexes but not 14 bp duplexes. Adenoviral VA RNA I, which inhibits dsRNA-dependent protein kinase (PKR), was tested as a fusion partner to express shRNA on the grounds that it might prevent nonspecific off-target effects. Fusion of VA RNA I to a lamin shRNA was moderately effective in silencing lamin expression, but gave strong OAS1 induction by an shRNA that does not induce OAS1 when expressed from the U6 or H1 promoters. To avoid interferon induction by U6 vectors, we recommend preserving the wild-type sequence around the transcription start site, in particular a C/G sequence at positions −1/+1, and we describe a simple cloning strategy using the Gateway recombination system that facilitates this task." @default.
• W1999601787 created "2016-06-24" @default.
• W1999601787 creator A5011134046 @default.
• W1999601787 creator A5061778211 @default.
• W1999601787 date "2004-03-01" @default.
• W1999601787 modified "2023-09-26" @default.
• W1999601787 title "Determinants of interferon-stimulated gene induction by RNAi vectors" @default.
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• W1999601787 doi "https://doi.org/10.1111/j.1432-0436.2004.07202001.x" @default.
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