Using ES cell-mediated transgenesis, we generated a novel mouse strain that permits a temporally and spatially controlled expression of responder genes in embryonic and multiple adult tissues. The transgene was constructed in a way that a CMV enhancer linked to the chicken β-actin promoter (CAG) drives the expression of the tetracycline-controlled transactivator (tTA) in particular tissues upon Cre-mediated excision of a floxed βgeo marker located between the promoter and the tTA. Based on the enzymatic activity of lacZ, the CAG-βgeo-tTA construct exhibits a widespread expression and appears to be very strong in the brain, heart, muscle, pancreas, and skin. Like the embryonic stem cell line that was used to generate this strain, the CAG-βgeo-tTA transgene is already highly active in preimplantation embryos. Using in vivo bioluminescence imaging on MMTV-Cre, CAG-βgeo-tTA, TetO-Luciferase triple transgenic mice and their controls, we demonstrated that the expression of the tTA, which is strictly dependent on the presence of Cre recombinase, induces the activation of the reporter transgene in the absence of any ligands. The tTA-mediated transactivation can be completely ablated through administration of doxycycline, and its subsequent withdrawal lifts the transcriptional block. Based on these characteristics, this novel strain may be useful in experiments that require a sustained expression of transgenes in particular cell types over a prolonged period followed by a rapid downregulation, for example in studies that examine the therapeutic value of cancer-initiating oncogenes during disease progression.