title: Adenoviral vectors expressing siRNAs for discovery and validation of gene function.
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, 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.
authors.affiliation: Galapagos Genomics BV, 2333 CN Leiden, The Netherlands.
authors.forename: Gert-Jan
authors.initials: GJ
authors.lastname: Arts
authors.forename: Ellen
authors.initials: E
authors.lastname: Langemeijer
authors.forename: Rudi
authors.initials: R
authors.lastname: Tissingh
authors.forename: Libin
authors.initials: L
authors.lastname: Ma
authors.forename: Heidi
authors.initials: H
authors.lastname: Pavliska
authors.forename: Kristina
authors.initials: K
authors.lastname: Dokic
authors.forename: Richele
authors.initials: R
authors.lastname: Dooijes
authors.forename: Emir
authors.initials: E
authors.lastname: Mesić
authors.forename: Remko
authors.initials: R
authors.lastname: Clasen
authors.forename: Frits
authors.initials: F
authors.lastname: Michiels
authors.forename: Jan
authors.initials: J
authors.lastname: van der Schueren
authors.forename: Mark
authors.initials: M
authors.lastname: Lambrecht
authors.forename: Sofie
authors.initials: S
authors.lastname: Herman
authors.forename: Reginald
authors.initials: R
authors.lastname: Brys
authors.forename: Kim
authors.initials: K
authors.lastname: Thys
authors.forename: Marcel
authors.initials: M
authors.lastname: Hoffmann
authors.forename: Peter
authors.initials: P
authors.lastname: Tomme
authors.forename: Helmuth
authors.initials: H
authors.lastname: van Es
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000328:Adult; D001172:Arthritis, Rheumatoid / Q000473:pathology; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000821:virology; D000078404:Epidermal Cells; D005347:Fibroblasts / Q000166:cytology / Q000473:pathology / Q000821:virology; D005786:Gene Expression Regulation / Q000235:genetics; D005796:Genes / Q000502:physiology*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology*; D015894:Genome, Human; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry / Q000821:virology; D009690:Nucleic Acid Conformation; D034741:RNA, Small Interfering / Q000096:biosynthesis* / Q000737:chemistry / Q000502:physiology*; D013329:Structure-Activity Relationship; D013583:Synovial Membrane / Q000473:pathology; D014162:Transfection; D014471:Umbilical Veins
chemical_list: D004279:DNA, Viral; D034741:RNA, Small Interfering