Figure 2.

Gata-6 overexpression in stem cells leads to endoderm differentiation and blocks stem cell states. (A) An inducible Gata-6 transgene integration by supertargeting. For diagnosis of the supertargeting event, the selected clones were analyzed by Southern blot hybridization as reported previously (Niwa et al. 1998). A 3.2-kb SacI fragment was detected with a probe from the 5′ end of lacZ, indicative of the replacement of the Oct-3/4 cDNA with Gata-6. Original ZHTc6 cells retained the 4.8-kb fragment of the preexisting Oct-3/4 transgene. Supertargeting events had taken place in all clones tested, and SKG612 is representing one of such clones. (B) Western blot analysis of SKG612 embyonic stem (ES) cells using anti-Gata-6 or anti-Gata-4 antibody after incubation for 24 h at the indicated concentrations of tetracycline (Tc). Induced expression of Gata-6 and Gata-4 protein was detected in SKG612 cells. (C) Colony morphology of SKG612 cells cultured for 4 d in the presence of leukemia inhibitory factor (LIF) and absence of Tc. (D) Electron micrograph of differentiated SKG612 cells. Magnification, ×4000. (E) Time course for the activation of endoderm associated genes and the repression of stem associated genes during the differentiation of SKG612 cells, analyzed by Northern blot. (F) Time course for the activation of endoderm associated genes and the repression of Bmp-4, analyzed by RT–PCR.