Transfection of a human LCRγ-globin gene into the EKLF−/− ES clones. A 26-kb Sal I fragment containing a human LCR/γ-globin construct and a PGK-puromycin resistance gene were coelectroporated into EKLF−/− ES cells. Three clones (2-4, 6-10, and 6-13) out of 27 puromycin-resistant clones carried the human LCR/γ-globin hybrid gene. The top panel shows a Southern blot analysis of Mlu I, Mlu I/Xho I, and HindIII restricted genomic DNAs using the indicated probe. The presence of a 15-kb Mlu I/Xho I and an overlapping 5-kb HindIII fragment in all three clones suggested at least 16 kb of the hybrid gene had integrated into the genome of the ES cells. The human LCR/γ-globin gene is shown at the bottom. The three solid black boxes represent the three exons of the human γ-globin gene and the solid gray box represents the probe used in the Southern blot analysis.