title: Viral vectors expressing a single microRNA-based short-hairpin RNA result in potent gene silencing in vitro and in vivo.
abstract: The characterization of RNA interference and the accompanying microRNAs (miRs), together with the exogenous expression of artificial miR-like elements, has led to the development of strategies for specific and potent gene silencing. In turn, this allows manipulation of gene expression levels for target validation purposes in cell culture or for the generation of animal models. In this study we determined the optimal strategy to achieve the most potent knockdown using miR-based viral vectors. We studied polycistronic miRs in a viral vector context and evaluated knockdown potency of multiple-miRs targeting the same seed sequence in parallel with miRs targeting different seed sequences, both for a reporter and endogenous mRNA targets. We demonstrate that potent knockdown can be obtained in vitro and in vivo using viral vectors that encode a single miR-based short-hairpin RNA and report a generic and effective cloning platform for artificial miR30-based short-hairpin RNAs to generate potent knockdown viral vectors.
authors.affiliation: KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium.
authors.forename: Luísa
authors.initials: L
authors.lastname: Osório
authors.affiliation: KU Leuven, Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium; KU Leuven, Leuven Viral Vector Core, Leuven, Belgium.
authors.forename: Rik
authors.initials: R
authors.lastname: Gijsbers
authors.affiliation: KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium.
authors.forename: Marusela
authors.initials: M
authors.lastname: Oliveras-Salvá
authors.affiliation: KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium; KU Leuven, Leuven Viral Vector Core, Leuven, Belgium.
authors.forename: Annelies
authors.initials: A
authors.lastname: Michiels
authors.affiliation: KU Leuven, Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, Leuven, Belgium.
authors.forename: Zeger
authors.initials: Z
authors.lastname: Debyser
authors.affiliation: KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium.
authors.forename: Chris
authors.initials: C
authors.lastname: Van den Haute
authors.affiliation: KU Leuven, Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, Leuven, Belgium. Electronic address: Veerle.Baekelandt@med.kuleuven.be.
authors.forename: Veerle
authors.initials: V
authors.lastname: Baekelandt
mesh_terms: D000818:Animals; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism; D005434:Flow Cytometry; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*
chemical_list: D035683:MicroRNAs; D034741:RNA, Small Interfering
keywords: Adeno-associated viral vector; Dopaminergic neurons; Lentiviral vector; Multiple hairpins; RNA interference; miR30