Fig. 1.
Fra-2 gain-of-function approach. (a) Transgenic construct for ectopic expression of fra-2 in vivo consisting of an H2Kb promoter, the genomic fra-2 locus, a reporter IRES-EGFP sequence, and a LTR sequence harboring a polyadenylation signal (pA). E1–E4 are Exons 1–4 of fra-2; HindIII (H) restriction sites and probe location (P) used for Southern blot analysis are indicated. (b) Southern blot analysis of tail DNA from wild-type mice (wt) and three independent fra-2tg (tg) lines (12, 13, and 15) by using the probe shown in a. (c) Kaplan–Meier plot showing increased mortality of transgenic lines 12 and 13. Mortality was not increased in line 15, which did not express the transgene. (d) Lungs of fra-2tg mice (tg, line 13, 20 weeks of age, n > 20) were increased in size. (e) Lung/body weight ratios of fra-2tg (tg, line 13) mice were increased at 15 weeks of age (n ≥ 3 mice per time point). (f) RNase-protection assay demonstrating fra-2 transgene expression in lungs of adult fra-2tg mice (line 13). gapdh was used as loading control. (g) Western blot analysis demonstrating ectopic expression of Fra-2 protein in the lungs of adult fra-2tg mice (line 13). The protein band pattern is characteristic for Fra-2. Actin was used as loading control. (h and i) Direct fluorescence of lung sections demonstrating the cellular localization of transgene expression. (h) Only background signal was observed in wt controls. (i) Transgene-encoded EGFP-fluorescence was detectable in neointimal cells (long arrow), peribronchial cells and VSMCs (short arrows), and fibroblastic cells (arrowheads) of fra-2tg mice (line 13, n = 3). Nuclei were counterstained with DAPI. (j) An adjacent section was stained with H&E to allow a better assessment of the structures indicated in i. A, artery; B, bronchus.