Generation of an embryonic/fetal lung-specific Tbx4-rtTA transgenic mouse line. (A) Schematic diagram of transgenic DNA construct. The position of the DNA probe for Southern blot is indicated. (B) Screen of transgenic founder lines by genomic DNA PCR. (C) Verification of transgenic lines by Southern blot. Genomic DNA was digested by XhoI and SalI prior to separation by gel electrophoresis. (D) Expression of rtTA-IRES-LacZ transgene in E13.5 lung was verified by X-gal staining (blue). (E) Cre-mediated mGFP expression (green) was detected in the lung of the triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG), but not in the double transgenic control mice (TetO-Cre/mT-mG), in which floxed-mTomato expression (red) was not affected. Dox induction was started from E6.5, and E13.5 mouse embryos were isolated and visualized under a fluorescence dissecting microscope. (F) Sagittal frozen section of E10.5 embryo of the triple transgenic mice with Dox induction from E6.5 to E10.5. Right panel shows the lung bud structure under high magnification. (G) Fluorescence microscopic examination of tissue frozen sections from E18.5 triple transgenic mice (Tbx4-rtTA/TetO-Cre/mT-mG), in which Dox induction was initiated from E6.5. mGFP was detected only in lung and tracheal mesenchyme, but not in other tissues. Dox, doxycycline; E, embryonic day.