Establishment of ΔmHS−25/6 and ΔmHS−3.5ΔmHS−25/6 ES cells. The Gata1 locus is schematically depicted with mutations at various steps of the single and double knockout establishments. mHS−25/6 was replaced in wild-type (A) and in previously established ΔmHS−3.5 ES cells (B)¹⁹  by a LoxP (black triangles)–flanked neomycin gene. The neomycin gene was excised by transient Cre expression. Locations of restriction sites for EcoRV (EV) and XbaI (X) are shown. P1 and P2 show the location recognized by the probes used for Southern blots. On the right, examples of Southern blots confirming the various mutations. Numbers above the blots correspond with the loci on the left. Size markers on the left are in kilobases, and the numbers in the blots show the expected size of the detected DNA fragments.