An improved tet-on system for gene expression in neurons delivered by a single lentiviral vector.
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.
Tian
Xuebi
X
Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Wang
Gongming
G
Xu
Ying
Y
Wang
Ping
P
Chen
Shasha
S
Yang
Hui
H
Gao
Feng
F
Xu
Aijun
A
Cao
Fei
F
Jin
Xiaogao
X
Manyande
Anne
A
Tian
Yuke
Y
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*
D011500:Protein Synthesis Inhibitors; D008156:Luciferases; D010751:Phosphopyruvate Hydratase; D013752:Tetracycline