Generation of inducible ES cells directing Smad1 and eGFP expression. (A) iSmad1iresEGFP ES cells were generated using the approach described in Kyba et al.²¹  Cre-mediated recombination of the targeting vector into the lox site on the X chromosome restores G-418 (NEO) resistance leading to the isolation of transgenic cells capable of tet-on induced sustained transgene expression. (B) Genomic PCR analysis of iSmad1 ES cells demonstrates site-specific transgene integration. Ainv-GFP is an iGFP line and positive control. The asterisk indicates a 500-bp PCR product that is generated only by a correctly integrated transgene. (C) FACS analysis of iGFP and iSmad1iresEGFP ES cell lines showing responsiveness to 1 μg/mL doxycycline. (D) An anti-Flag Western blot of extracts from iSmad1iresEGFP cells untreated (iSmad1) or exposed for 8 hours to 1 μg/mL doxycycline (+dox). A Smad1⁺ control sample (first lane) along with extract from Ainv parental ES cells (second lane) are also included. (E) An anti-Flag Western blot of extracts made from day 6 embryoid body samples that were induced at different times of development. Once added, the induction was maintained in the EBs until day 6 as described in “Materials and methods.” In all samples EBs were cultured until day 6 when they were harvested and processed by Western blotting. (F) Western blot analysis of cytoplasmic and nuclear extracts from untreated (no dox) and iSmad1 EBs that were induced with doxycycline at day 2 and harvested at day 3 (1 μg/mL dox). For each sample, 30 μg protein lysate was analyzed with the antibodies indicated on the right side of the panel. Note that panel F is composed of 4 different panel strips from top to bottom; each independent strip represents the same samples that were analyzed with the different antisera. Nucleoporin and Bcl-2 are nuclear and cytoplasmic markers, respectively, which indicates that the enriched separation of nuclear and cytoplasmic compartments was successful. The lane marked Fl-Smad1 is a positive control whole cell extract generated by transient transfection of a flag-tagged Smad1 expression vector.