1. Adenoviral Vectors Expressing siRNAs for Discovery and Validation of Gene Function
- Author
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Mark Jacques Yvonne Lambrecht, Emir Mesic, Remko Johannes Clasen, Rudi Tissingh, Kim Thys, Peter Herwig Maria Tomme, Heidi Pavliska, Marcel Hoffmann, Libin Ma, Gert-Jan Arts, Kristina Dokic, Jan van der Schueren, Helmuth van Es, Sofie Herman, Frits Michiels, Richele Dooijes, Ellen Langemeijer, and Reginald Christophe Xavier Brys
- Subjects
Adult ,Keratinocytes ,Umbilical Veins ,Small interfering RNA ,Genetic Vectors ,Computational biology ,Biology ,Transfection ,Adenoviridae ,Cell Line ,Arthritis, Rheumatoid ,Structure-Activity Relationship ,Transduction (genetics) ,Plasmid ,RNA interference ,Gene expression ,Methods ,Genetics ,Humans ,RNA, Small Interfering ,Gene ,Genetics (clinical) ,Regulation of gene expression ,Genome, Human ,Synovial Membrane ,Fibroblasts ,Epidermal Cells ,Gene Expression Regulation ,Genes ,DNA, Viral ,Nucleic Acid Conformation ,Endothelium, Vascular - Abstract
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, GαS, 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.
- Published
- 2003