Design and cloning of lentiviral vectors expressing small interfering RNAs

RNA interference ( RNAi) has emerged as a powerful technique to downregulate gene expression. The use of polIII promoters to express small hairpin RNAs ( shRNAs), combined with the versatility and robustness of lentiviral vector - mediated gene delivery to a wide range of cell types offers the possibility of long- term downregulation of specific target genes both in vitro and in vivo. The use of silencing lentivectors allows for a rapid and convenient way of establishing cell lines ( or transgenic mice) that stably express shRNAs for analysis of phenotypes produced by knockdown of a gene product. Here we present two possible protocols describing the design and cloning of silencing lentiviral vectors. These protocols can be completed in less than 3 weeks.