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dc.contributor.authorFrank, Sander B.
dc.contributor.authorSchulz, Veronique V.
dc.contributor.authorMiranti, Cindy K.
dc.date.accessioned2017-04-27T23:38:13Z
dc.date.available2017-04-27T23:38:13Z
dc.date.issued2017-02-28
dc.identifier.citationA streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector 2017, 17 (1) BMC Biotechnologyen
dc.identifier.issn1472-6750
dc.identifier.pmid28245848
dc.identifier.doi10.1186/s12896-017-0341-x
dc.identifier.urihttp://hdl.handle.net/10150/623280
dc.description.abstractBackground: 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.
dc.description.sponsorshipNational Cancer Institute of the National Institutes of Health [R01CA154835, P30CA023074]; Department of Defense [W81XWH-14-1-0479]; Worldwide Cancer Fund [11-0082]; Van Andel Research Institute; University of Arizonaen
dc.language.isoenen
dc.publisherBIOMED CENTRAL LTDen
dc.relation.urlhttp://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-017-0341-xen
dc.rights© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0.en
dc.rights.urihttps://creativecommons.org/licenses/by/4.0/
dc.subjectpLKOen
dc.subjectshRNAen
dc.subjectLentivirusen
dc.subjectInducibleen
dc.titleA streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vectoren
dc.typeArticleen
dc.contributor.departmentUniv Arizona, Ctr Canc, Dept Cellular & Mol Meden
dc.identifier.journalBMC Biotechnologyen
dc.description.noteOpen Access Journalen
dc.description.collectioninformationThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.en
dc.eprint.versionFinal published versionen
refterms.dateFOA2018-06-25T05:38:52Z
html.description.abstractBackground: 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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© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0.
Except where otherwise noted, this item's license is described as © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0.