THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2006; 8: 433–441. Published online 3 January 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.860 Stable RNA interference: comparison of U6 and H1 promoters in endothelial cells and in mouse brain Petri I. M¨ akinen 1 Jonna K. Koponen 1† Anna-Mari K¨ arkk¨ ainen 1† Tarja M. Malm 2 Kati H. Pulkkinen 1 Jari Koistinaho 2 Mikko P. Turunen 1 Seppo Yl¨ a-Herttuala 1,3,4 * 1 Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland 2 Department of Neurobiology, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland 3 Department of Medicine, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland 4 Gene Therapy Unit, Kuopio University Hospital, Puijonlaaksontie 2, FIN-70210 Kuopio, Finland *Correspondence to: Seppo Yl¨ a-Herttuala, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. E-mail: seppo.ylaherttuala@uku.fi † These authors contributed equally to this work. Received: 15 April 2005 Revised: 19 September 2005 Accepted: 22 September 2005 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. Keywords RNAi; siRNA; lentiviral vector; U6 promoter; H1 promoter; gene therapy Introduction RNA interference (RNAi) is a biological, strongly conserved post- transcriptional gene silencing mechanism mediated by double-stranded RNA molecules (dsRNAs) (for review, see [1]). It was first found and applied in Caenorhabditis elegans [2], but since the breakthrough discovery that RNAi can also be exploited in mammalian cells [3], it has become a useful tool in biological studies (for review, see [4]). Synthetic small interfering RNA molecules (siRNAs) are used in many gene function studies, but their use for gene therapy of most mono- or multigenic diseases is limited, because the introduction of synthetic siRNAs leads only to a short-term gene silencing. Thus, plasmid and viral vectors expressing short hairpin RNAs (shRNAs) have been developed for transient or long-term Copyright  2006 John Wiley & Sons, Ltd.