Fig. 2

Characterization of doxycycline-inducible Wnt4 transgenic mice. (A) Luciferase reporter gene expression in tet-Wnt4/MMTV-rtTA transgenic mammary glands. To express Wnt4 specifically in mammary glands, tet-Wnt4A and B mice were mated with transgenic mice that express rtTA under the control of MMTV-LTR promoter (MMTV-rtTA). The subsequent virgin female littermates (3 months old) were administered doxycycline containing water (2 mg/ml) for 1 month. Mammary gland lysates were prepared, and the luciferase activity of 10 μg of total protein lysate was measured. N.T, non-transgenic; rtTA, MMTV-rtTA; W4A, tet-Wnt4A strain; W4B, independent strain tet-Wnt4B; bitransgenics are shown as W4A or B/rtTA. (B) Induction of Wnt4 mRNA expression in mammary glands in doxycycline-administered mice. Wnt4 mRNA level was quantified by real time quantitative PCR (see methods) using total RNAs from mammary glands of mice administered doxycycline for 1 month. The fold induction is indicated with respect to control glands (from 7 week old mice). For mRNA preparations from non, single and bi-transgenic glands, plus designations indicate the presence of the transgene, and the order is shown as follows, Wnt4A/rtTA. Data marked with an asterix were samples from mice in estrus. (C) Induction of Wnt4 protein expression in doxycycline-treated bitransgenic mammary epithelial cells. Mammary epithelial cells were prepared from non-transgenic (-/-) and bitransgenic (+/+) glands and cultured for 2 days. Cells were treated with doxycycline (1 μg/ml) for another 24 hrs and lysates were prepared for Western blot analysis. Lysates from expression vector-transfected 293T cells were used as positive controls.