FIG. 2.

Isolation of ES clones deleted for both sir2α genes. R1 ES cells (lane 1) that had homologously integrated the hygromycin resistance vector (lane 2) were electroporated with a second knockout construct carrying the lacZ-neomycin resistance (β-geo) gene (35), and the cells were selected for resistance to G418. Among the clones isolated, some, like that shown in lane 3, had the β-geo sequence in place of the hygromycin resistance-thymidine kinase sequence, while others, like that shown in lane 4, had both the β-geo and the hygromycin resistance-thymidine kinase sequences homologously integrated into the two sir2α genes. The Southern blot shown in the lower panel was derived from DNA digested with NsiI plus BamHI and probed with the sequence shown, derived from DNA outside the region used for the knockout vectors.