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Fig. 3. The inhibitory effect of Smad7 is reversible. (A) Strategy for generation of fSmad7-10⁺ and fSmad7-10^- ES cells. fSmad7-10⁺ was generated by stable integration of floxed-Smad7 cDNA transgene under the CAG promoter into EB3 ES cells. After elctroporation of pCAGGS-Cre into fSmad7-10⁺, fSmad7-10^- was free of Smad7 cDNA and expressed DsRed. (B) Colony morphologies of fSmad7-10⁺ (left) and Smad7-10^- (right) cells. EB3 (control), fSmad7-10⁺ and fSmad7-10^- ES cells were seeded at 2000 cells/well in six-well plates, and cultured for 1 week in the medium supplemented with puromycin. The lower panel shows expression of DsRed. (C) Colony morphologies of fSmad7-10⁺, fSmad7-10^-, and control ES cells. fSmad7-10⁺ (middle), fSmad7-10^- (right) and EB3 (as control, left) ES cells were cultured in FCS-containing medium for 5 days. Numbers indicate numbers of colonies appearing (n=3). The lower panel shows AP staining. Bars, 5 mm (upper panels); 200 µm (lower panels). (D) Relative numbers of each transfectant present on Days 1, 3, and 5 of culture compared with Day 0. Each bar represents the mean ± s.e.m. (n=4). (E) Relative proliferation intensity is shown as total cell number on Day 5/number of colonies appearing, compared with that of EB3 ES cells (100%). Each bar represents the mean ± s.e.m. (n=3). (F) Expression of stem cell marker genes Oct3-4, Sox2, and Rex-1 in fSmad7-10⁺, fSmad7-10^- and EB3 (as control) ES cells examined by northern blot analysis. ES cells were cultured in FCS-containing medium for 5 days. Gapdh was detected as a loading control. (G) Chimeric mice derived from fSmad7-10^- cells that had been cultured for at least 1 month. Chimeric mice (upper panel) expressed DsRed, whereas control mice (lower panel) exhibited no DsRed fluorescence.

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