Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells.
This article describes the first (to our knowledge) tetracycline-inducible regulatory system that demonstrates that the tetracycline repressor (tetR) alone, rather than tetR-mammalian cell transcription factor fusion derivatives, can function as a potent trans-modulator to regulate gene expression in mammalian cells. With proper positioning of tetracycline operators downstream of the TATA element and of human epidermal growth factor (hEGF) as a reporter, we show that gene expression from the tetracycline operator-bearing hCMV major immediate-early enhancer-promoter (pcmvtetO) can be regulated by tetR over three orders of magnitude in response to tetracycline when (1) the reporter was cotransfected with tetR-expressing plasmid in transient expression assays, and (2) the reporter unit was stably integrated into the chromosome of a tetR-expressing cell line. This level of tetR-mediated inducible gene regulation is significantly higher than that of other repression-based mammalian cell transcription switch systems. In an in vivo porcine wound model, close to 60-fold tetR-mediated regulatory effects were detected and it was reversed when tetracycline was administered. Collectively, this study provides a direct implementation of this tetracycline-inducible regulatory switch for controlling gene expression in vitro, in vivo, and in gene therapy.
Yao
F
F
Division of Plastic Surgery, Brigham and Women's Hospital, Boston, MA 02115, USA.
Svensjö
T
T
Winkler
T
T
Lu
M
M
Eriksson
C
C
Eriksson
E
E
D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics; D004815:Epidermal Growth Factor / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics; D018014:Gene Transfer Techniques; D017781:Genes, Immediate-Early / Q000235:genetics; D017930:Genes, Reporter; D006367:HeLa Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics*; D012516:Osteosarcoma; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics* / Q000502:physiology; D013552:Swine; D016385:TATA Box / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D014947:Wounds and Injuries / Q000235:genetics
D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D004815:Epidermal Growth Factor; D013752:Tetracycline