Figure 1.
Generation of SPC-Cre-ERT2 mice.
A) Schematic map of SPC-Cre-ERT2 expression cassette. HSPC-P, human surfactant protein C promoter; Cre-ERT2, Cre coding sequence fused with a tamoxifen-inducible estrogen receptor. pA, a polyA sequence from SV40 virus. Cre-F primer binding sites, 561–579 bp of Cre-ERT2 transgene; Cre-R primer binding sites, 976–997 bp of Cre-ERT2 transgene. The map is drawn in scale. B) Screening SPC-Cre-ERT2 transgenic mice using PCR. Genomic DNA from each mouse tail was used as template to specifically PCR-amplify the Cre-ERT2 transgene. M, DNA marker; +, SPC-Cre-ERT2 plasmid DNA control; -, water control; F8, F13, F16, F42, F67 are five representative founder transgenic mice generated by microinjection of SPC-Cre-ERT2 expression cassette into fertilized embryos. C) Cre-ERT2 fusion proteins were detected using Western blot in lung tissues of SPC-Cre-ERT2 transgenic mice. Lane 1, C57BL/6J mouse; Lane 2, an offspring of F42 founder without Cre-ERT2 transgene when genotyped using PCR; Lane 3, 4, 5, offspring of F42 founder; Lane 6, 7, 8, offspring of F67 founder. Notice the variable expression levels of Cre-ERT2 in offspring from the same founder.
Figure 2.
Tamoxifen-inducible and tissue-specific recombinase activity of Cre-ERT2 in SPC-Cre-ERT2/ROSA26R transgenic mice.
A) β-galactosidase activity was detected in the lung alveolar epithelia from a SPC-Cre-ERT2/ROSA26R transgenic mouse receiving tamoxifen treatment. a & b, lung tissue sections from ROSA26R mice (without Cre-ERT2 transgene) receiving vehicle (a) or tamoxifen (b); c & d, lung tissue sections from SPC-Cre-ERT2/ROSA26R transgenic mice receiving vehicle (c) or tamoxifen (d). B) Endogenous β-galactosidase activity was found in lung bronchial epithelia of ROSA26R mouse receiving vehicle (a) and SPC-Cre-ERT2/ROSA26R transgenic mouse after tamoxifen treatment (b). C) X-gal stained lung tissues of SPC-Cre-ERT2/ROSA26R transgenic mouse receiving tamoxifen were also immune-stained for proSP-C, an alveolar type II cell-specific marker. Arrows indicate three representative alveolar type II cells co-expressing proSP-C (brown) and β-galactosidase (blue). D) β-galactosidase activity was detected only in the lung alveolar epithelium, but not in other organs from a SPC-Cre-ERT2/ROSA26R transgenic mouse receiving tamoxifen treatment. a, heart; b, liver; c, kidney; d, intestine; e, lung. All scale bars in this figure equal 100 µm.
Figure 3.
Tamoxifen-induced recombinase activity of Cre-ERT2 in the lung tissues of SPC-Cre-ERT2/TSC1fx/fx transgenic mice.
DNAs from the lung tissues of SPC-Cre-ERT2/TSC1fx/fx transgenic mice treated with vehicle or tamoxifen were examined by PCR to detect TSC1 deletion as an indication of Cre-ERT2 recombinase activity. M, DNA marker; +, a transgenic mouse with TSC1 deletion; -, C57BL/6J mouse. Other lanes, offspring from F42 and F67 founders treated with vehicle (vehi) or tamoxifen (tam).