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Methods

Adenoviral Vectors Expressing siRNAs for Discovery and Validation of Gene Function

Gert-Jan Arts¹, Ellen Langemeijer¹, Rudi Tissingh¹, Libin Ma¹, Heidi Pavliska¹, Kristina Dokic¹, Richele Dooijes¹, Emir Mei¹, Remko Clasen¹, Frits Michiels¹, Jan van der Schueren², Mark Lambrecht², Sofie Herman², Reginald Brys², Kim Thys², Marcel Hoffmann¹, Peter Tomme² and Helmuth van Es^1,3

¹ Galapagos Genomics BV, 2333 CN Leiden, The Netherlands ² Galapagos Genomics NV, B-2800 Mechelen, Belgium

RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GS, we have measured inhibition of ligand-dependant, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.1332603. Article published online before print in September 2003.

[Supplemental Material is available online at www.genome.org.]

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