Adenoviral vectors expressing siRNAs for discovery and validation of gene function

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 tran...

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Bibliographic Details
Published in:	Genome research Vol. 13; no. 10; pp. 2325 - 2332
Main Authors:	Arts, Gert-Jan, Langemeijer, Ellen, Tissingh, Rudi, Ma, Libin, Pavliska, Heidi, Dokic, Kristina, Dooijes, Richele, Mesić, Emir, Clasen, Remko, Michiels, Frits, van der Schueren, Jan, Lambrecht, Mark, Herman, Sofie, Brys, Reginald, Thys, Kim, Hoffmann, Marcel, Tomme, Peter, van Es, Helmuth
Format:	Journal Article
Language:	English
Published:	United States Cold Spring Harbor Laboratory Press 01-10-2003
Subjects:	Adenoviridae - genetics Adult Arthritis, Rheumatoid - pathology Cell Line DNA, Viral - genetics Endothelium, Vascular - chemistry Endothelium, Vascular - cytology Endothelium, Vascular - virology Epidermis - cytology Fibroblasts - cytology Fibroblasts - pathology Fibroblasts - virology Gene Expression Regulation - genetics Genes - physiology Genetic Vectors - biosynthesis Genetic Vectors - chemistry Genetic Vectors - physiology Genome, Human Humans Keratinocytes - chemistry Keratinocytes - virology Methods Nucleic Acid Conformation RNA, Small Interfering - biosynthesis RNA, Small Interfering - chemistry RNA, Small Interfering - physiology Structure-Activity Relationship Synovial Membrane - pathology Transfection Umbilical Veins
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Summary:	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, GalphaS, we have measured inhibition of ligand-dependent, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Corresponding author. E-MAIL es@galapagos.be ; FAX (31) 71 5248 801.
ISSN:	1088-9051
DOI:	10.1101/gr.1332603