98. Stable Lentivirus-Mediated RNA Interference: Comparison of U6 and H1 Promoters in Endothelial Cells and GFP Knockdown in Mouse Brain

Abstract

RNA interference (RNAi) is a post-transcriptional RNA degradation process, which has become a useful tool in gene function studies and recently in gene therapy applications. Long-term expression of short hairpin RNA molecules (shRNA) required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the shRNA expression are the human U6 small nuclear promoter (U6) and human H1 promoter (H1). Our goal was to construct an efficient and optimal LV-based shRNA expression system and to explore LV-mediated RNAi for long-term gene silencing. For this purpose, we systematically compared the U6 and H1 promoters for optimal silencing of green fluorescent protein (GFP) in mouse endothelial cells. We show that the U6 promoter is more efficient than H1 in GFP silencing, leading to 80% GFP knockdown at the average of one integrated vector genome per target cell genome. In addition, the silencing was persistent for several months. Finally, as a proof of principle, we demonstrate that the GFP expression is silenced in vivo after stereotactic LV injection into GFP transgenic mice brain. In conclusion, LV-mediated RNAi is a powerful gene silencing method, which is useful for gene therapy applications requiring long-term knockdown of gene functions in vitro and in vivo.