Plasmid-Based RNA Interference: Construction of Small-Hairpin RNA Expression Vectors

Recent work demonstrates that RNA interference (RNAi) helps coordinate the flow of information from transcription to protein expression, complicating tremendously our former understanding of how protein expression is regulated (1 ,2 ). Inhibitory RNAs can be expressed naturally in cells as microRNAs (miRNAs) or introduced into cells as small, inhibitory RNAs (siRNAs). Both processes inhibit gene expression, but by different mechanisms. In general, miRNAs inhibit translation. One cellular process that benefits from “paused” translation is the nerve synapse (3 ). siRNAs inhibit expression through targeted degradation of target mRNA. Both miRNAs and siRNAs can be used at the bench to silence mRNA expression. In this chapter, we provide a practical approach to accomplish siRNA-mediated gene silencing, through the generation and introduction of plasmids expressing short-hairpin RNAs (shRNAs) that are subsequently processed to siRNAs in vivo.