Fig. 1. Characterization of transgene and determination of MnSOD transcription in vivo.

(A) MnSOD promoter–enhancer-driven luciferase expression construct for the generation of transgenic mice. The 2.7 kb human MnSOD basal promoter- and enhancer-driven luciferase gene construct was used for microinjection to generate transgenic mice. (B) a, Detection of human MnSOD gene in transgenic mice. Kpn1 and BglII restriction-digested genomic DNAs from mouse tails were electrophoresed in 1% agarose gel, transferred onto nylon membrane and then hybridized with MnSOD intronic element. Transgenic mice showed ExI₂E band at ~2.7 kb. Endogenous genes were detected in both transgenic and non-transgenic animals; b, the luciferase activity was determined from the skin tissue extracts in transgenic mice and their non-transgenic littermates using luciferase assay kit (Promega); c, MnSOD protein and d, activity levels in transgenic mice and their nontransgenic littermates. (C) Bioluminescence images of mice treated with TPA (4 µg/mouse/day) were acquired by CCD camera. (D) Bioluminescent signals were quantified in terms of photon count which in turn represents the reporter gene activity in living mice. Bioluminescent images were gated to an area (1.54 cm²) where signals are generated after the application of substrate d-luciferin. Photon counts were estimated within that defined gated area. (E) The same animals were humanely euthanized and skin tissues were harvested. The luciferase activity was determined from the skin tissue extracts using luciferase assay kit (Promega). (F) MnSOD activity (U/mg protein) in controls and TPA-treated skin tissues. All data are representative of three independent sets of experiments. Significantly different from control; *P < 0.05 and **P < 0.01; significantly different from 4-day repeated treatment groups; #P < 0.01.