Abstract
Although RNA interference (RNAi) is a popular technique, no method for simultaneous silencing of multiple targets by small-hairpin RNA (shRNA)-expressing RNAi vectors has yet been established. Although gene silencing can be achieved by synthetic small-interfering RNA (siRNA) duplexes, the approach is transient and largely dependent on the transfection efficiency of the host cell. We offer a solution: a simple, restriction enzyme-generated stable RNAi technique that can efficiently silence multiple targets with a single RNAi vector and a single selection marker. In this study, we succeeded in simultaneous stable knockdown of transforming growth factor beta (TGF-beta) pathway-related Smads--Smad2, Smad3 and Smad4--at the cellular level. We observed distinct phenotypic changes in TGF-beta-dependent cellular functions such as invasion, wound healing and apoptosis. This method is best suited for an analysis of complex signal transduction pathways in which silencing of a single gene cannot account for the whole process.