In one construct (Tnnt2-rtTA), a 0.5-kb rat Tnnt2 promoter was inserted in front of a nuclear-localized (nls) rtTA cDNA followed by an elongation factor-1 (EF1) polyadenylation signal. The Tnnt2-rtTA fragment was flanked by mouse H19 insulator sequences to prevent ectopic transcription influences from random insertion sites. The second construct (tetO-cre) was generated by cloning the nls-cre sequence downstream of the tetO-CMV minimal promoter fragment. Purified constructs were coinjected into fertilized FVB/N oocytes. Seven double-transgenic founders contained both constructs and transmitted them in Mendelian fashion indicating that both constructs were inserted at the same site. One line demonstrated reproducible recombination of the R26R reporter after doxycycline treatment and was propagated and maintained.