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Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies
RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences s...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9043282/ https://www.ncbi.nlm.nih.gov/pubmed/35492592 http://dx.doi.org/10.3389/fphys.2022.836106 |
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author | Verdonckt, Thomas-Wolf Vanden Broeck, Jozef |
author_facet | Verdonckt, Thomas-Wolf Vanden Broeck, Jozef |
author_sort | Verdonckt, Thomas-Wolf |
collection | PubMed |
description | RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the E. coli HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for in vitro experiments. In this paper, we describe and compare methods for the economical (43.2 €/mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the Helicoverpa-specific gene Dicer2 and, as a non-endogenous control, the Green Fluorescent Protein gene (GFP). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates. |
format | Online Article Text |
id | pubmed-9043282 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-90432822022-04-28 Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies Verdonckt, Thomas-Wolf Vanden Broeck, Jozef Front Physiol Physiology RNA interference (RNAi) is a highly conserved pathway for the post-transcriptional regulation of gene expression. It has become a crucial tool in life science research, with promising potential for pest-management applications. To induce an RNAi response, long double-stranded RNA (dsRNA) sequences specific to the target gene must be delivered to the cells. This dsRNA substrate is then processed to small RNA (sRNA) fragments that direct the silencing response. A major obstacle to applying this technique is the need to produce sufficiently large amounts of dsRNA in a very cost-effective manner. To overcome this issue, much attention has been given to the development and optimization of biological production systems. One such system is the E. coli HT115 strain transformed with the L4440 vector. While its effectiveness at inducing knockdowns in animals through feeding of the bacteria has been demonstrated, there is only limited knowledge on the applicability of bacteria-derived dsRNA for in vitro experiments. In this paper, we describe and compare methods for the economical (43.2 €/mg) and large-scale (mg range) production of high-quality dsRNA from the HT115 bacterial system. We transformed the bacteria with constructs targeting the Helicoverpa-specific gene Dicer2 and, as a non-endogenous control, the Green Fluorescent Protein gene (GFP). First, we compared the total RNA extraction yields of four cell-lysis treatments: heating, lysozyme digestion, sonication, and a control protocol. Second, we assessed the quality and purity of these extracted dsRNAs. Third, we compared methods for the further purification of dsRNAs from crude RNA extracts. Finally, we demonstrated the efficiency of the produced dsRNAs at inducing knockdowns in a lepidopteran cell line. The insights and results from this paper will empower researchers to conduct otherwise prohibitively expensive knockdown studies, and greatly reduce the production times of routinely or large-scale utilized dsRNA substrates. Frontiers Media S.A. 2022-04-13 /pmc/articles/PMC9043282/ /pubmed/35492592 http://dx.doi.org/10.3389/fphys.2022.836106 Text en Copyright © 2022 Verdonckt and Vanden Broeck. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Physiology Verdonckt, Thomas-Wolf Vanden Broeck, Jozef Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies |
title | Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies |
title_full | Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies |
title_fullStr | Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies |
title_full_unstemmed | Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies |
title_short | Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies |
title_sort | methods for the cost-effective production of bacteria-derived double-stranded rna for in vitro knockdown studies |
topic | Physiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9043282/ https://www.ncbi.nlm.nih.gov/pubmed/35492592 http://dx.doi.org/10.3389/fphys.2022.836106 |
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