Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells.
The doxycycline (dox)-inducible Tet-On system is widely used to control gene expression in mammalian cells. This system is based on the bacterial Tet operon, which has been modified and improved for its function in eukaryotic cells. To identify the optimal system for different applications, we compared Tet-On variants in frequently used cell types that were either transiently transfected with the relevant plasmids or stably transduced with an "all-in-one" lentiviral vector. The V10 variant performed optimally in the transiently transfected cells and demonstrated no background activity without dox, high dox-induced activity and the highest fold-induction. Because of its very high dox-sensitivity, the V16 system may be preferred if only low intracellular dox concentrations can be reached. V16 performed optimally in the transduced cells and demonstrated the highest activity and dox-sensitivity without background activity. Moreover, V16 demonstrated more robust induction of gene expression after a latency period without dox. This study provides important findings for choosing the optimal Tet-On system for diverse cell culture settings. V10 is the best system for most applications in which the DNA is episomally present in cells, whereas V16 may be optimal when the Tet-On components are stably integrated in the cellular genome.
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. a.t.das@amc.uva.nl.
Atze T
AT
Das
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Xue
X
Zhou
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Stefan W
SW
Metz
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Monique A
MA
Vink
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Ben
B
Berkhout
D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D009876:Operon; D012097:Repressor Proteins / Q000235:genetics*; D014161:Transduction, Genetic; D014162:Transfection
D001426:Bacterial Proteins; D002352:Carrier Proteins; D012097:Repressor Proteins; C083139:Tet O resistance protein, Bacteria; C034811:tetracycline resistance-encoding transposon repressor protein; D004318:Doxycycline
Doxycycline; Gene expression; Tet-On system; Transcription regulation; rtTA