pSM155 and pSM30 vectors for miRNA and shRNA expression.

MicroRNAs (miRNAs) have key roles in diverse regulatory pathways, including control of developmental timing, cell differentiation, apoptosis, cell proliferation, and organ development. miRNAs regulate gene function through a process termed RNA interference (RNAi), which is a highly conserved intracellular mechanism that regulates posttranscriptional gene silencing. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs (shRNAs) generated from an expression vector. In some recently described vectors, the siRNAs are expressed from synthetic stem-loop precursors of microRNAs (miRNAs) driven by polymerase II promoters. We have reported new RNAi vectors, designated pSM155 and pSM30, that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors, these pSM155 and pSM30 constructs can efficiently downregulate the expression of their target genes. Moreover, the expression of a coexpressed fluorescent marker, EGFP, is substantially improved by this new design. The new vectors can also be used to express natural miRNAs and label cells expressing these miRNAs. These RNAi vectors thus provide new tools for gene suppression and miRNA expression. We describe in this chapter the protocols for selecting and cloning artificial and natural miRNAs (or shRNAs), evaluating their efficiency in downregulating gene expression, and also discuss the potential applications of these vectors.