Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines
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 limit...
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , |
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| Format: | Journal Article |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10356/101612 http://hdl.handle.net/10220/18735 |
| _version_ | 1826122662438502400 |
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| author | Börner, Kathleen Niopek, Dominik Cotugno, Gabriella Kaldenbach, Michaela Pankert, Teresa Willemsen, Joschka Zhang, Xian Schürmann, Nina Streetz, Konrad Kräusslich, Hans-Georg Mockenhaupt, Stefan Serva, Andrius Grimm, Dirk Hiet, Marie-Sophie Wiedtke, Ellen Binder, Marco Castoldi, Mirco Starkuviene, Vytaute Erfle, Holger Bartenschlager, Ralf Boutros, Michael Gilbert, Daniel F. |
| author2 | School of Biological Sciences |
| author_facet | School of Biological Sciences Börner, Kathleen Niopek, Dominik Cotugno, Gabriella Kaldenbach, Michaela Pankert, Teresa Willemsen, Joschka Zhang, Xian Schürmann, Nina Streetz, Konrad Kräusslich, Hans-Georg Mockenhaupt, Stefan Serva, Andrius Grimm, Dirk Hiet, Marie-Sophie Wiedtke, Ellen Binder, Marco Castoldi, Mirco Starkuviene, Vytaute Erfle, Holger Bartenschlager, Ralf Boutros, Michael Gilbert, Daniel F. |
| author_sort | Börner, Kathleen |
| collection | NTU |
| description | 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. |
| first_indexed | 2024-10-01T05:51:50Z |
| format | Journal Article |
| id | ntu-10356/101612 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T05:51:50Z |
| publishDate | 2014 |
| record_format | dspace |
| spelling | ntu-10356/1016122023-02-28T17:05:03Z Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines Börner, Kathleen Niopek, Dominik Cotugno, Gabriella Kaldenbach, Michaela Pankert, Teresa Willemsen, Joschka Zhang, Xian Schürmann, Nina Streetz, Konrad Kräusslich, Hans-Georg Mockenhaupt, Stefan Serva, Andrius Grimm, Dirk Hiet, Marie-Sophie Wiedtke, Ellen Binder, Marco Castoldi, Mirco Starkuviene, Vytaute Erfle, Holger Bartenschlager, Ralf Boutros, Michael Gilbert, Daniel F. School of Biological Sciences DRNTU::Science::Biological sciences 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. Published version 2014-01-29T02:11:59Z 2019-12-06T20:41:26Z 2014-01-29T02:11:59Z 2019-12-06T20:41:26Z 2013 2013 Journal Article Börner, 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. https://hdl.handle.net/10356/101612 http://hdl.handle.net/10220/18735 10.1093/nar/gkt836 24049077 en Nucleic acids research © 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. application/pdf |
| spellingShingle | DRNTU::Science::Biological sciences Börner, Kathleen Niopek, Dominik Cotugno, Gabriella Kaldenbach, Michaela Pankert, Teresa Willemsen, Joschka Zhang, Xian Schürmann, Nina Streetz, Konrad Kräusslich, Hans-Georg Mockenhaupt, Stefan Serva, Andrius Grimm, Dirk Hiet, Marie-Sophie Wiedtke, Ellen Binder, Marco Castoldi, Mirco Starkuviene, Vytaute Erfle, Holger Bartenschlager, Ralf Boutros, Michael Gilbert, Daniel F. Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines |
| title | Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines |
| title_full | Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines |
| title_fullStr | Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines |
| title_full_unstemmed | Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines |
| title_short | Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines |
| title_sort | robust rnai enhancement via human argonaute 2 overexpression from plasmids viral vectors and cell lines |
| topic | DRNTU::Science::Biological sciences |
| url | https://hdl.handle.net/10356/101612 http://hdl.handle.net/10220/18735 |
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