Establishment of TET-G1KO cells. (A) Targeted GATA-1 allele. Schematic maps of the Gata-1 locus, the targeting vector, and the targeted locus are shown from top to bottom, respectively. Exon numbers are indicated. Restriction enzyme cleavage sites are shown as follows: K, KpnI; E, EcoRI; X, XbaI; B, BstEII. (B) Southern blot analysis. Homologous recombination was detected by Southern blot analysis of EcoRI-digested genomic DNA using a probe between 2 EcoRI sites of GATA-1 cDNA, which included exons 3-6. Homologous recombination brought about the band of 4.7 kb (open arrow head) instead of the 2.9-kb band (filled arrow head) in the control cells. (C) Construct of GATA-1 (HRES) EGFP. TetO stands for the tetracycline-responsive element. (D-E) Fluorescence-activated cell-sorting (FACS) analysis of EGFP and Western blot of GATA-1 expression in ES cells induced by the deprivation of tetracycline. (F-G) No effects are seen of the GATA-1–null mutation on myeloid differentiation of ES cells. Control and TET-G1KO ES cells were differentiated in the presence of 100 ng/mL tetracycline into macrophages (F) or granulocytes (G) in the presence of M-CSF or GM-CSF, respectively. May-Giemsa staining and FACS analysis of myeloid cell surface markers are shown in the top and bottom panels, respectively.