title: Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness.
abstract: BACKGROUND
  The tetracycline-regulatable system is one of the most valuable tools for controlling gene expression. In its current form, however, the system is less than ideal for in vivo or gene therapy uses due to difficulties in set-up procedures, high basal leakiness, and unpredictable delivery and efficiency.
  
  
  METHODS
  To address these issues, we have devised a second generation of tetracycline-regulated promoters (TREs). The second-generation TRE (SG-TRE) contains a shortened cytomegalovirus (CMV) minimal promoter together with eight tet operator sequences positioned in an optimized manner upstream of the TATA box. This construct displays far greater reduction in basal leakiness than maximal transgene expression. Conversely, maximal transgene expression is increased to a greater degree than basal leakiness by post-translational stabilization with bovine growth hormone poly A.
  
  
  RESULTS
  In transient studies, the SG-TRE displays over 100 000-fold regulation efficiency in HeLa cells at 1:1 ratio of transactivator to reporter plasmid in the Tet-Off system. This novel promoter achieves a regulation efficiency 500- to 1000-fold higher than that of the original TRE (P(hCMV*-1)) in HeLa cells by displaying undetectable levels of basal leakiness without compromised maximal expression. In other cell lines, the SG-TRE proves to be more efficient than the original P(hCMV*-1) in a cell-dependent manner. Furthermore, the SG-TRE preserves its enhanced regulation efficiency and its reduced basal leakiness in the context of a single positive feedback regulatory vector that presents ease of delivery of the system for use in vivo. Finally, in vivo, the biological function of granulocyte-macrophage colony stimulating factor is tightly regulated in the context of SG-TRE delivered via adeno-associated viruses.
authors.affiliation: Hillman Cancer Center, University of Pittsburgh Medical Center, Pittsburgh, PA 15261, USA. mohammadis@msx.upmc.edu
authors.forename: Siamak
authors.initials: S
authors.lastname: Agha-Mohammadi
authors.forename: Mark
authors.initials: M
authors.lastname: O'Malley
authors.forename: Abrak
authors.initials: A
authors.lastname: Etemad
authors.forename: Zhong
authors.initials: Z
authors.lastname: Wang
authors.forename: Xiao
authors.initials: X
authors.lastname: Xiao
authors.forename: Michael T
authors.initials: MT
authors.lastname: Lotze
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D006801:Humans; D009875:Operator Regions, Genetic; D011401:Promoter Regions, Genetic; D013752:Tetracycline / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013752:Tetracycline