miRNA and shRNA Expression Vectors Based on mRNA and miRNA Processing

RNA interference (RNAi) has emerged as a powerful tool in basic research and therapeutics by silencing the expression of specific target genes. RNAi is triggered by double-stranded small interfering RNAs, which can be processed from naturally or artificially expressed microRNAs (miRNAs) or small hairpin RNAs (shRNAs). The development of reliable RNAi vectors encoding artificial and natural miRNAs would be useful tools for basic research and therapeutic applications. We reported a new type of new RNAi vectors, designated pSM155, and pSM30 that take into consideration of miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. These vectors significantly improved the expression of an enhanced green fluorescent protein marker from the same mRNA transcript and also provide a simplified cloning method. We have described in this chapter the protocols for cloning artificial and natural miRNAs (or shRNAs), evaluating their efficiency in downregulating gene expression, and also discuss the potential applications of these vectors.