Narendra V. Sankpal, Timothy P. Fleming, William E. Gillanders

RNA interference (RNAi) is a powerful new tool for the selective ablation of gene expression, facilitating loss-of-function studies. However, appropriate controls are considered essential to confirm the specificity of RNAi experiments. The most stringent control is rescue of the target gene in a form that is refractory to RNAi. To facilitate rescue of the target gene, we have created improved dual expression lentiviral vectors with the ability to simultaneously drive expression of a shRNA for RNA interference and a rescue transgene in a single vector system. In proof-of-principle experiments, we ablated more than 90% of target gene expression by targeting either the open reading frame, or the 3' UTR region. Target gene expression was successfully rescued with a cDNA containing silent third-codon point mutations in the targeted region or with native cDNA when the 3' UTR was targeted. Finally, expression of the rescue transgene can be manipulated by positional cloning and appropriate promoter selection. The dual expression lentiviral vectors described here represent a versatile strategy for confirming the integrity of RNAi experiments and may facilitate functional analyses even in the absence of an established gain-of-function model system.