Adenovirus-delivered antisense RNA and shRNA exhibit different silencing efficiencies for the endogenous transforming growth factor-β (TGF-β) type II receptor

Gene silencing is an essential tool in gene discovery and gene therapy. Traditionally, viral delivery of antisense RNA and, more recently, small interfering RNA (siRNA) molecules in the form of small hairpin RNAs (shRNA) has been used as a strategy to achieve gene silencing. Nevertheless, the enduring challenge is to identify molecules that specifically and optimally silence a given target gene. In this study, we tested a set of adenovirus-delivered antisense RNA fragments and adenovirus-delivered shRNA molecules for their ability to target human transforming growth factor-beta type II receptor (TGF beta RII). We used a dicistronic reporter, consisting of the coding sequences for TGF beta RII and green fluorescent protein (GFP) to screen for optimal silencing agents targeting TGF beta RII. Our results show, for both antisense RNA and shRNA molecules, that their effectiveness in the GFP screen correlated directly with their ability to reduce exogenously expressed TGF beta RII. Unexpectedly, the antisense RNAs were unable to silence endogenous TGF beta RII. In contrast, the shRNAs were able to silence endogenous TGF beta RII. The shRNA that demonstrated the most pronounced effect on the dicistronic TGF beta RII/GFP reporter reduced endogenous TGF beta RII protein expression by 70% in A549 cells and reduced TGF beta signaling by > 80% in HeLa cells.