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.
abstract: 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.
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Chambers
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: William P
authors.initials: WP
authors.lastname: Halford
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Joshua
authors.initials: J
authors.lastname: Geltz
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Olga
authors.initials: O
authors.lastname: Villamizar
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Gross
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Alison
authors.initials: A
authors.lastname: Embalabala
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Edward
authors.initials: E
authors.lastname: Gershburg
authors.affiliation: Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.
authors.forename: Andrew
authors.initials: A
authors.lastname: Wilber
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D004251:DNA Transposable Elements; D004318:Doxycycline / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D015534:Trans-Activators; D013752:Tetracycline; D004318:Doxycycline
references.citation: Cell. 1997 Nov 14;91(4):501-10
references.pmid: 9390559
references.citation: Proc Natl Acad Sci U S A. 1996 May 28;93(11):5185-90
references.pmid: 8643550
references.citation: Mol Ther. 2006 Mar;13(3):625-30
references.pmid: 16368272
references.citation: J Mol Biol. 2002 Oct 25;323(3):441-52
references.pmid: 12381300
references.citation: Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3665-70
references.pmid: 21307310
references.citation: Expert Opin Biol Ther. 2005 May;5(5):627-38
references.pmid: 15934839
references.citation: Cell. 2013 Aug 15;154(4):914-27
references.pmid: 23953119
references.citation: Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547-51
references.pmid: 1319065
references.citation: Gene. 1991 Dec 15;108(2):193-9
references.pmid: 1660837
references.citation: Methods Enzymol. 2000;327:401-21
references.pmid: 11044999
references.citation: Hum Gene Ther. 2003 Feb 10;14(3):277-97
references.pmid: 12639307
references.citation: Cell. 2002 Jun 28;109(7):807-9
references.pmid: 12110178
references.citation: J Virol. 1993 Dec;67(12):7373-82
references.pmid: 7901428
references.citation: PLoS One. 2010;5(6):e10975
references.pmid: 20544015
references.citation: Nucleic Acids Res. 1995 Sep 11;23(17):3605-6
references.pmid: 7567477
references.citation: Nat Genet. 2009 Jun;41(6):753-61
references.pmid: 19412179
references.citation: Hum Gene Ther. 2002 May 20;13(8):979-88
references.pmid: 12031130
references.citation: J Mol Genet Med. 2006 Jul 17;2(1):107-18
references.pmid: 19565004
references.citation: Nucleic Acids Res. 2001 Aug 15;29(16):3327-34
references.pmid: 11504870
references.citation: Nat Biotechnol. 2005 Jan;23(1):108-16
references.pmid: 15619618
references.citation: Curr Gene Ther. 2004 Mar;4(1):15-31
references.pmid: 15032611
references.citation: Mol Cell Biol. 1986 Mar;6(3):792-800
references.pmid: 3022130
references.citation: J Virol. 1986 Feb;57(2):629-37
references.pmid: 3003394
references.citation: Int J Biochem Mol Biol. 2013 Dec 15;4(4):201-8
references.pmid: 24380024
references.citation: Mol Ther. 2005 Sep;12(3):569-74
references.pmid: 15964244
references.citation: J Mol Biol. 2002 May 17;318(5):1221-35
references.pmid: 12083513
references.citation: PLoS One. 2014;9(11):e112712
references.pmid: 25390293
references.citation: Biochim Biophys Acta. 2012 Jul;1819(7):688-93
references.pmid: 22366276
references.citation: Biochemistry (Mosc). 2013 Apr;78(4):335-41
references.pmid: 23590436
references.citation: J Virol. 2001 Jul;75(13):6143-53
references.pmid: 11390616
references.citation: Biotechnol Appl Biochem. 1988 Feb;10(1):6-12
references.pmid: 2841949
references.citation: Proc Natl Acad Sci U S A. 2002 Mar 19;99(6):3740-5
references.pmid: 11891299
references.citation: Gene Ther. 2008 Mar;15(5):384-90
references.pmid: 18283290
references.citation: Gene Ther. 2010 Mar;17(3):380-8
references.pmid: 19907502
references.citation: In Silico Biol. 2003;3(3):235-40
references.pmid: 12954087
references.citation: BMC Biotechnol. 2010;10:81
references.pmid: 21106052