Fig. 1. Generation of the Pax6 ectoderm enhancer null allele. (A) Schematic representation of the Pax6 ectoderm enhancer targeted deletion strategy. The purple box represents the 341 bp Pax6 ectoderm enhancer. The regions of sequence used as probes to assess the targeting procedure are pictured as white boxes. The 2.7 kb 5' targeting arm is represented by the yellow box, and the 3.6 kb 3' targeting arm is represented by the orange box. The loxP site-specific recombination sequences for cre recombinase are indicated by white triangles. The sizes of the restriction fragments detected by the probes are indicated by lines located above the corresponding map. Small arrows indicate the location of primer pairs (primers P1, P2 and P3) used for PCR genotyping. The sizes of the PCR products are indicated above the primer pairs. (B) Southern blotting to identify wild-type (+/+) and targeted (where +/– designates +/neoEE) ES cell-line genomic DNA for EcoRI and SphI restriction digests probed with the 5' and 3' probes, respectively. The fragment sizes are labeled next to the appropriate bands. (C) PCR genotyping of genomic DNA. The sizes of PCR products are indicated to the left and right of the gel panel. R, EcoRI; N, NcoI; Sa, SacII; Sp, SphI; A, AatII; K, KpnI; neo, neomycin phosphotransferase gene; tk, thymidine kinase gene; pA, polyadenylation signal; Pr, promoter.