Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines.

Börner K, Niopek D, Cotugno G, Kaldenbach M, Pankert T, Willemsen J, Zhang X, Schürmann N, Mockenhaupt S, Serva A, Hiet M, Wiedtke E, Castoldi M, Starkuviene V, Erfle H, Gilbert D, Bartenschlager R, Boutros M, Binder M, Streetz K, Kräusslich H, Grimm D (2013)

Publication Status: Published

Publication Type: Journal article, Original article

Publication year: 2013

Journal

Nucleic Acids Research Oxford University Press

Book Volume: 41

Pages Range: e199

Journal Issue: 21

DOI: 10.1093/nar/gkt836

Abstract

As 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.

Authors with CRIS profile

Daniel Gilbert Lehrstuhl für Medizinische Biotechnologie

Involved external institutions

Universitätsklinikum Heidelberg DE Germany (DE) Universitätsklinikum Aachen (UKA) DE Germany (DE) Ruprecht-Karls-Universität Heidelberg DE Germany (DE) Deutsches Krebsforschungszentrum (DKFZ) DE Germany (DE)

How to cite

APA:

Börner, K., Niopek, D., Cotugno, G., Kaldenbach, M., Pankert, T., Willemsen, J.,... Grimm, D. (2013). Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines. Nucleic Acids Research, 41(21), e199. https://doi.org/10.1093/nar/gkt836

MLA:

Börner, K, et al. "Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines." Nucleic Acids Research 41.21 (2013): e199.

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