AbstractBackgroundViral vectors have been used in several different settings for the delivery of small hairpin (sh) RNAs. However, most vectors have utilized ubiquitously‐expressing polymerase (pol) III promoters to drive expression of the hairpin as a result of the strict requirement for precise transcriptional initiation and termination. Recently, pol II promoters have been used to construct vectors for RNA interference (RNAi). By embedding the shRNA into a micro RNA‐context (miRNA) the endogenous miRNA processing machinery is exploited to achieve the mature synthetic miRNA (smiRNA), thereby expanding the possible promoter choices and eventually allowing cell type specific down‐regulation of target genes.MethodsIn the present study, we constructed lentiviral vectors expressing smiRNAs under the control of pol II promoters to knockdown gene expression in cell culture and in the brain.ResultsWe demonstrate robust knockdown of green fluorescent protein using lentiviral vectors driving RNAi from the ubiquitously‐expressing promoter of the cytomegalovirus (CMV) and, in addition, we show for the first time neuron‐specific knockdown in the brain using a neuron‐specific promoter. Furthermore, we show that the expression pattern of the presumed ubiquitously‐expressing CMV promoter changes over time from being expressed initially in neurons and glial cells to being expressed almost exclusively in neurons in later stages.ConclusionsIn the present study, we developed vectors for cell‐specific RNAi for use in the brain. This offers the possibility of specifically targeting RNAi to a subset of cells in a complex tissue and may prove to be of great importance in the design of future gene therapeutic paradigms. Copyright © 2009 John Wiley & Sons, Ltd.