Protocol

Design and Cloning of an shRNA into a Lentiviral Silencing Vector: Version B

This protocol was adapted from "Development of Lentiviral Vectors Expressing siRNA," Chapter 3, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.

INTRODUCTION

This protocol describes the use of lentiviral vectors to deliver small interfering RNA (siRNA)-mediated silencing cassettes. The combination of these two technologies allows for the development of a powerful tool to achieve long-term down-regulation of specific target genes both in vitro and in vivo. It combines the specificity of RNA interference with the versatility of lentiviral vectors to stably transduce a wide range of cell types. In this method, a small hairpin (shRNA) is cloned initially into an entry vector (pENTR/U6) immediately downstream from an hU6 promoter. The silencing cassette is flanked by recombination sites from bacteriophage (attL1 and attL2). Once an effective shRNA is obtained, it can be transferred to the destination vector. The destination vector is a lentiviral vector carrying a marker (green fluorescent protein [GFP] or a selection marker) with a destination cassette cloned upstream of the marker (attR1 and attR2 flanking a ccdB toxic gene). Thus, the silencing cassette can be transferred from the entry vector to the destination vector in a simple Gateway LR cloning reaction. The positioning of the silencing cassette upstream of the marker expression cassette avoids down-regulation of the marker.