Inclusion of a matrix-attached region in a 7SK pol III vector increases the efficiency of shRNA-mediated gene silencing in embryonic carcinoma cells.

ABSTRACT

RNA interference is a widely used tool for analysis of gene function in mammalian cells. Stable knockdown of specific target genes can be maintained in cell lines and live organisms using vector-based delivery of short hairpins (shRNAs) driven by RNA polymerase III promoters. Here we describe a vector incorporating the human 7SK promoter for shRNA-mediated gene silencing in the P19 embryonic carcinoma stem cell line. Our preliminary experiments with the 7SK shRNA expression vector indicated that its activity could be hindered by random genomic integration. In order to counter this inhibitory mechanism, we inserted a matrix-attached region sequence to generate an episomal vector system. We compared the effects of insertion versus exclusion of the MAR sequence on the shRNA-mediated gene-specific silencing of the beta-tubulin III and Cyclophilin A genes. While the MAR sequence is not strongly correlated with the episomal status of the expression vector, our studies indicate that inclusion of the MAR element significantly enhances the stability of shRNA-mediated gene silencing in the P19 stem cells.