How to make tetracycline-regulated transgene expression go on and off.
Tetracycline-regulated gene expression systems are widely used to allow temporal and quantitative control of transgene expression in cultured cells and transgenic animals. While working with the Tet-Off system, where tetracycline or the analogue doxycycline suppresses expression, we noted a considerable variability in induced transgene expression after removal of doxycycline. Variable expression of the transgene could not be explained by clonal variation since it was noted when working with clonal cell lines. Instead we found that doxycycline bound nonspecifically to cells and extracellular matrix and was slowly released after it had been removed from tissue culture media. The released doxycycline reached sufficiently high levels to completely suppress transgene expression. The effect was not dependent on cell type or the nature of the transgene. However, robust and rapid transgene expression could be induced if released doxycycline were removed by washing cells 3h after the initial removal of doxycycline. The use of different vector systems, harboring the tetracycline-regulatable components, yielded similar results. These results not only help explain why tetracycline-regulatable transgene expression systems sometimes are variable but also provide simple ways to substantially improve the efficiency, utility, and reliability of these widely used expression systems.
Rennel
Emma
E
The Rudbeck Laboratory, Vascular Biology Unit, Department of Genetics and Pathology, Uppsala University, S-751 85 Uppsala, Sweden.
Gerwins
Pär
P
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology; D002460:Cell Line; D004318:Doxycycline / Q000493:pharmacokinetics / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors / Q000187:drug effects; D005910:Glioma / Q000378:metabolism; D007700:Kinetics; D015203:Reproducibility of Results; D013752:Tetracycline / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects; D019076:Transgenes; D014407:Tumor Cells, Cultured
D013752:Tetracycline; D004318:Doxycycline