A system for creating stable cell lines that express a gene of interest from a bidirectional and regulatable herpes simplex virus type 1 promoter
Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. We envisione...
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| Veröffentlicht in: | PloS one 2015-03, Vol.10 (3), p.e0122253 |
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| creator | Chambers, Christopher B Halford, William P Geltz, Joshua Villamizar, Olga Gross, Jeffrey Embalabala, Alison Gershburg, Edward Wilber, Andrew |
| description | Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. We envisioned that the ideal system should be tightly controlled and coupled with the ability to efficiently create and identify stable cell lines. Herein, we describe a system based upon a bidirectional Herpes simplex virus type 1 promoter that is naturally responsive to the VP16 transactivator and modified to permit tetracycline-regulated transcription on one side while maintaining constitutive activity on the other side. Incorporation of this element into the Sleeping Beauty transposon resulted in a novel bidirectional system with the capacity for high-efficiency stable integration. Using this system, we created stable cell lines in which expression of a gene of interest was tightly and uniformly controlled across a broad range of levels via a novel combination of doxycycline-sensitive de-repression and VP16-mediated sequence-specific induction. The unique characteristics of this system address major limitations of current methods and provide an excellent strategy to investigate the effects of gene dosing in mammalian models. |
| doi_str_mv | 10.1371/journal.pone.0122253 |
| format | Article |
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We envisioned that the ideal system should be tightly controlled and coupled with the ability to efficiently create and identify stable cell lines. Herein, we describe a system based upon a bidirectional Herpes simplex virus type 1 promoter that is naturally responsive to the VP16 transactivator and modified to permit tetracycline-regulated transcription on one side while maintaining constitutive activity on the other side. Incorporation of this element into the Sleeping Beauty transposon resulted in a novel bidirectional system with the capacity for high-efficiency stable integration. Using this system, we created stable cell lines in which expression of a gene of interest was tightly and uniformly controlled across a broad range of levels via a novel combination of doxycycline-sensitive de-repression and VP16-mediated sequence-specific induction. 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This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Chambers et al 2015 Chambers et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-9bdd0069bfc5c9f606905b23d811e66f6b30d19e278f62e5eee7b6b3a0011d703</citedby><cites>FETCH-LOGICAL-c692t-9bdd0069bfc5c9f606905b23d811e66f6b30d19e278f62e5eee7b6b3a0011d703</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378986/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378986/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2926,23864,27922,27923,53789,53791,79370,79371</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25823013$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Kues, Wilfried A.</contributor><creatorcontrib>Chambers, Christopher B</creatorcontrib><creatorcontrib>Halford, William P</creatorcontrib><creatorcontrib>Geltz, Joshua</creatorcontrib><creatorcontrib>Villamizar, Olga</creatorcontrib><creatorcontrib>Gross, Jeffrey</creatorcontrib><creatorcontrib>Embalabala, Alison</creatorcontrib><creatorcontrib>Gershburg, Edward</creatorcontrib><creatorcontrib>Wilber, Andrew</creatorcontrib><title>A system for creating stable cell lines that express a gene of interest from a bidirectional and regulatable herpes simplex virus type 1 promoter</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. 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The unique characteristics of this system address major limitations of current methods and provide an excellent strategy to investigate the effects of gene dosing in mammalian models.</description><subject>Antibiotics</subject><subject>Biology</subject><subject>Biotechnology</subject><subject>Cell Line</subject><subject>Cell Line, Tumor</subject><subject>Cell lines</subject><subject>Chromosomes</subject><subject>Cloning</subject><subject>DNA Transposable Elements</subject><subject>Doxycycline</subject><subject>Doxycycline - pharmacology</subject><subject>Drug resistance</subject><subject>Gene expression</subject><subject>Gene Expression - drug effects</subject><subject>Gene Expression - genetics</subject><subject>Gene Expression Regulation, Viral - drug effects</subject><subject>Gene Expression Regulation, Viral - genetics</subject><subject>Genes</subject><subject>Genetic engineering</subject><subject>Genomes</subject><subject>HEK293 Cells</subject><subject>HeLa Cells</subject><subject>Herpes simplex</subject><subject>Herpes simplex virus</subject><subject>Herpes viruses</subject><subject>Herpesvirus 1, Human - 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We envisioned that the ideal system should be tightly controlled and coupled with the ability to efficiently create and identify stable cell lines. Herein, we describe a system based upon a bidirectional Herpes simplex virus type 1 promoter that is naturally responsive to the VP16 transactivator and modified to permit tetracycline-regulated transcription on one side while maintaining constitutive activity on the other side. Incorporation of this element into the Sleeping Beauty transposon resulted in a novel bidirectional system with the capacity for high-efficiency stable integration. Using this system, we created stable cell lines in which expression of a gene of interest was tightly and uniformly controlled across a broad range of levels via a novel combination of doxycycline-sensitive de-repression and VP16-mediated sequence-specific induction. The unique characteristics of this system address major limitations of current methods and provide an excellent strategy to investigate the effects of gene dosing in mammalian models.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25823013</pmid><doi>10.1371/journal.pone.0122253</doi><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Antibiotics Biology Biotechnology Cell Line Cell Line, Tumor Cell lines Chromosomes Cloning DNA Transposable Elements Doxycycline Doxycycline - pharmacology Drug resistance Gene expression Gene Expression - drug effects Gene Expression - genetics Gene Expression Regulation, Viral - drug effects Gene Expression Regulation, Viral - genetics Genes Genetic engineering Genomes HEK293 Cells HeLa Cells Herpes simplex Herpes simplex virus Herpes viruses Herpesvirus 1, Human - genetics Humans Immunology Integration Medicine Plasmids Promoter Regions, Genetic - drug effects Promoter Regions, Genetic - genetics Proteins Tetracycline - pharmacology Trans-Activators - pharmacology Transcription Transcription, Genetic - drug effects Transcription, Genetic - genetics Viruses VP16 protein |
| title | A system for creating stable cell lines that express a gene of interest from a bidirectional and regulatable herpes simplex virus type 1 promoter |
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