title: An improved tet-on system for gene expression in neurons delivered by a single lentiviral vector.
abstract: In most cases, the successful application of gene therapy requires the development of vectors that can provide regulated control of therapeutic gene expression. We have reconstituted the Tet-On (tetracycline-regulated transgene expression) two-component system in a single lentiviral vector with insertion of a chicken chromatin insulator (cHS4) element between the two expression cassettes. Optimization of this vector included an improved reverse tetracycline-dependent trans-activator (rtTA) sequence developed through HIV viral evolution, and an rtTA-dependent, Tet-responsive element containing modifications of the tetO sequence (TRE-tight1) to improve leaky basal transcription. Transduction of HeLa cells with these lentiviral vectors resulted in a high level of rtTA expression in the presence of doxycycline. In neuronal cells, rtTA expression driven by a neuron-specific enolase (NSE) promoter was more efficient than gene expression from a murine cytomegalovirus promoter. Transgene expression from the NSE promoter also provided tightly regulated gene expression in neurons in vivo.
authors.affiliation: Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
authors.forename: Xuebi
authors.initials: X
authors.lastname: Tian
authors.forename: Gongming
authors.initials: G
authors.lastname: Wang
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.forename: Shasha
authors.initials: S
authors.lastname: Chen
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Aijun
authors.initials: A
authors.lastname: Xu
authors.forename: Fei
authors.initials: F
authors.lastname: Cao
authors.forename: Xiaogao
authors.initials: X
authors.lastname: Jin
authors.forename: Anne
authors.initials: A
authors.lastname: Manyande
authors.forename: Yuke
authors.initials: Y
authors.lastname: Tian
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D023421:Models, Animal; D018146:Muromegalovirus / Q000235:genetics*; D009474:Neurons / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: D011500:Protein Synthesis Inhibitors; D008156:Luciferases; D010751:Phosphopyruvate Hydratase; D013752:Tetracycline