Introduced the reverse tetracycline transactivator (rtTA), enabling the Tet-On system. A mutant Tet repressor was fused to VP16 such that the resulting rtTA only binds tetO sequences in the presence of doxycycline—the opposite logic of the original tTA (Tet-Off). Addition of doxycycline to HeLa cells constitutively expressing rtTA induced a stably integrated reporter more than 1,000-fold. This inverted pharmacology (drug-on = gene-on) is operationally preferred for in vivo applications where baseline silencing is required without drug administration. The rtTA/TRE pair is the dominant inducible regulatory design used in AAV vectors requiring conditional or titratable transgene expression.
Transcriptional activation by tetracyclines in mammalian cells
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Key findings
Abstract
A transcriptional transactivator was developed that fuses the VP16 activation domain with a mutant Tet repressor from Escherichia coli. This transactivator requires certain tetracycline (Tc) derivatives for specific DNA binding. Thus, addition of doxycycline to HeLa cells that constitutively synthesized the transactivator and that contained an appropriate, stably integrated reporter unit rapidly induced gene expression more than a thousandfold. The specificity of the Tet repressor-operator-effector interaction and the pharmacological characteristics of Tc's make this regulatory system well suited for the control of gene activities in vivo, such as in transgenic animals and possibly in gene therapy.