dc.contributor.authorBörner, Kathleen
dc.contributor.authorNiopek, Dominik
dc.contributor.authorCotugno, Gabriella
dc.contributor.authorKaldenbach, Michaela
dc.contributor.authorPankert, Teresa
dc.contributor.authorWillemsen, Joschka
dc.contributor.authorZhang, Xian
dc.contributor.authorSchürmann, Nina
dc.contributor.authorMockenhaupt, Stefan
dc.contributor.authorServa, Andrius
dc.contributor.authorHiet, Marie-Sophie
dc.contributor.authorWiedtke, Ellen
dc.contributor.authorCastoldi, Mirco
dc.contributor.authorStarkuviene, Vytaute
dc.contributor.authorErfle, Holger
dc.contributor.authorGilbert, Daniel F.
dc.contributor.authorBartenschlager, Ralf
dc.contributor.authorBoutros, Michael
dc.contributor.authorBinder, Marco
dc.contributor.authorStreetz, Konrad
dc.contributor.authorKräusslich, Hans-Georg
dc.contributor.authorGrimm, Dirk
dc.date.accessioned2014-01-29T02:11:59Z
dc.date.available2014-01-29T02:11:59Z
dc.date.copyright2013en_US
dc.date.issued2013
dc.identifier.citationBörner, K., Niopek, D., Cotugno, G., Kaldenbach, M., Pankert, T., Willemsen, J., et al. (2013). Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines. Nucleic acids research, 41(21), e199.en_US
dc.identifier.urihttp://hdl.handle.net/10220/18735
dc.description.abstractAs the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.en_US
dc.language.isoenen_US
dc.relation.ispartofseriesNucleic acids researchen_US
dc.rights© 2013 The Authors. This paper was published in Nucleic Acids Research and is made available as an electronic reprint (preprint) with permission of The Authors. The paper can be found at the following official DOI: [http://dx.doi.org/10.1093/nar/gkt836].  One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.en_US
dc.subjectDRNTU::Science::Biological sciences
dc.titleRobust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell linesen_US
dc.typeJournal Article
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.identifier.doihttp://dx.doi.org/10.1093/nar/gkt836
dc.description.versionPublished versionen_US


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