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A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector

BACKGROUND: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermor...

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Autores principales: Frank, Sander B., Schulz, Veronique V., Miranti, Cindy K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331646/
https://www.ncbi.nlm.nih.gov/pubmed/28245848
http://dx.doi.org/10.1186/s12896-017-0341-x
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author Frank, Sander B.
Schulz, Veronique V.
Miranti, Cindy K.
author_facet Frank, Sander B.
Schulz, Veronique V.
Miranti, Cindy K.
author_sort Frank, Sander B.
collection PubMed
description BACKGROUND: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. METHODS: First, we modified the Tet-pLKO-Puro vector to make it easy (“EZ”) for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. RESULTS: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. CONCLUSIONS: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.
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spelling pubmed-53316462017-03-03 A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector Frank, Sander B. Schulz, Veronique V. Miranti, Cindy K. BMC Biotechnol Methodology Article BACKGROUND: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. METHODS: First, we modified the Tet-pLKO-Puro vector to make it easy (“EZ”) for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. RESULTS: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. CONCLUSIONS: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing. BioMed Central 2017-02-28 /pmc/articles/PMC5331646/ /pubmed/28245848 http://dx.doi.org/10.1186/s12896-017-0341-x Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Frank, Sander B.
Schulz, Veronique V.
Miranti, Cindy K.
A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector
title A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector
title_full A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector
title_fullStr A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector
title_full_unstemmed A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector
title_short A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector
title_sort streamlined method for the design and cloning of shrnas into an optimized dox-inducible lentiviral vector
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331646/
https://www.ncbi.nlm.nih.gov/pubmed/28245848
http://dx.doi.org/10.1186/s12896-017-0341-x
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