Tet-induced overexpression of Runx1 down-regulates Flk-1 in differentiating ES cells. (A) The construct and strategy of Tet-regulated expression of Runx1 in ES cells. The expression of Runx1 and IRES-linked GFP is driven by a Tet-regulated promoter suppressed by the addition of tetracycline (Tet-off system). (B-C) Inducible expression of GFP and Runx1 by the removal of Tet in undifferentiated ES cells. GFP expression was analyzed using FACS (B), and Runx1 expression was analyzed using Western blotting (C) 48 hours after Tet removal. (D-E) Inducible expression of Runx1 down-regulates Flk-1 expression during in vitro differentiation of ES cells. ES cells with Tet-inducible GFP constructs (top lane) or Tet-inducible Runx1-IRES-GFP constructs (bottom lane) were cultured on collagen type 4–coated dishes in the presence or absence of tetracycline and analyzed for Flk-1 and GFP expression by flow cytometry (D) and for Flk-1 expression by semiquantitative RT-PCR (E) on day 4.5 of differentiation. Results shown are representative of 3 independent experiments.