Homozygous CAG-stop-tTA2 transgenic mice are viable and fertile. CAG-stop-tTA2 transgenic mice harbor the ii-CAG-stop-tTA2-IRES-tau-lacZ-ii transgene; designed with a loxP-flanked transcriptional STOP cassette preventing transcription of the downstream modified tetracycline-regulated transactivator (tTA2). The ii-CAG-stop-tTA2-IRES-tau-lacZ-ii transgene is flanked by two copies of the chicken β-globin HS4 insulator on each side to preserve expression fidelity (see additional information below). When bred to mice that express a tamoxifen-inducible Cre recombinase (CreERT2), administration of tamoxifen to the double mutant offspring allows the CreERT2 fusion protein to enter the nucleus of the cre-expressing cells; this deletes the STOP cassette and results in expression of tTA2. The donating investigator reports that tau-lacZ fusion protein expression in the tamoxifen-treated double mutant offspring is faint. Of note, the donating investigator has not confirmed if breeding CAG-stop-tTA2 transgenic mice with mice expressing constitutively active Cre recombinase results in tTA2 expression in the double mutant offspring.

Applying both Cre-lox and Tet-Off technologies, these CAG-stop-tTA2 transgenic mice may be useful to generate compound mutant mice in which expression of a tetracycline-responsive promoter element (TRE or tetO)-driven gene of interest can be both directed to the cell types defined by the chosen Cre recombinase expression, as well as turned off by the addition of tetracycline (or its analog doxycycline [dox]).

These CAG-stop-tTA2 transgenic mice may be used to design a tripartite system to express fluorescently tagged synaptic proteins with both spatial and temporal control. When CAG-stop-tTA2 transgenic mice are bred with TRE-Bi-SG-T transgenic mice (Stock No. 012345), the resulting CAG-stop-tTA2::TRE-Bi-SG-T double mutant mice allow tdTomato and synaptophysin/GFP fusion protein expression to be determined by the tissue-specific tamoxifen-inducible Cre recombinase expression of a third strain of the researchers choosing. If such CAG-stop-tTA2::TRE-Bi-SG-T double mutant mice were bred with CaMKII-CreER mice (Stock No. 012362), the resulting tamoxifen-treated triple mutant mice should exhibit red-labeling of whole cortical/hippocampal/striatal neurons and green-labeling of their presynaptic terminals in the absence of dox. Similarly, if CAG-stop-tTA2::TRE-Bi-SG-T double mutant mice were bred with GAD2-CreER mice (Stock No. 010702), the resulting tamoxifen-treated triple mutant mice should exhibit red-labeling of whole GABAergic interneurons and green-labeling of their presynaptic terminals in the absence of dox.

The CAG-stop-tTA2 transgene is flanked by chicken β-globin HS4 insulators. The insulators are reported to preserve expression fidelity from transgene integration/position effects by blocking expansion of heterochromatin into the transgene (potentially preventing it from being silenced), as well as decrease the basal expression/increase the inducibility of TREs.