Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain

✍ Scribed by Petri I. Mäkinen; Jonna K. Koponen; Anna-Mari Kärkkäinen; Tarja M. Malm; Kati H. Pulkkinen; Jari Koistinaho; Mikko P. Turunen; Seppo Ylä-Herttuala

• Publisher: John Wiley and Sons
• Year: 2006
• Tongue: English
• Weight: 536 KB
• Volume: 8
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.860

✦ Synopsis

Abstract

Background

RNA interference (RNAi) is a post‐transcriptional RNA degradation process, which has become a very useful tool in gene function studies and gene therapy applications. Long‐term cellular expression of small interfering RNA (siRNA) molecules required for many gene therapy applications can be achieved by lentiviral vectors (LVs). The two most commonly used promoters to drive the short hairpin RNA (shRNA) expression are the human U6 small nuclear promoter (U6) and the human H1 promoter (H1).

Methods

We investigated whether there is any significant difference between the efficiencies of U6 and H1 in LV‐mediated RNAi using green fluorescent protein (GFP) as a target gene by flow cytometry and real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) in endothelial cells. Also, we compared the efficiencies of U6 and H1 in the GFP transgenic mouse brain after stereotactic LV injection.

Results

We show that the U6 promoter is more efficient than H1 in GFP silencing in vitro, leading to 80% GFP knockdown at an average of one integrated vector genome per target cell genome. The silencing is persistent for several months. In addition, the U6 promoter is superior to H1 in vivo and leads to stable GFP knockdown in mouse brain for at least 9 months.

Conclusions

These results show that LV‐mediated RNAi is a powerful gene‐silencing method for the long‐term inhibition of gene expression in vitro and in vivo. Copyright © 2006 John Wiley & Sons, Ltd.