Fig. 2. Gene targeting strategy to generate the Pitx2 c allele. (A) Summary of exon use by Pitx2 isoforms. (B) Pitx2 genomic structure and Pitx2c-specific targeting strategy. Boxes represent exons and straight lines introns. P1 and P2 indicate two promoters that regulate expression of different isoforms. (C) Pitx2 c targeted allele before and after PGKneomycin removal. At the bottom, the Pitx2 null allele (abc^null) and cre knock-in alleles (abc^creneo) also used in this study are shown. (D) Southern blot with flanking probes: left panel shows a Southern blot of tail DNA probed with 5' flanking probe and center panel shows Southern blot probed with 3' flanking probe. Right panel shows a Southern blot probed with 3' flanking probe after crossing to CMV cre recombinase deletor strain to generate Pitx2 c^+/- mice. After recombination, the 3' flanking probe hybridizes to an 8 kb fragment and a 10 kb fragment in mice retaining PGKneomycin. The `+' above lanes denotes mice retaining PGKneomycin and `-' indicates a mouse that deleted PGKneomycin. (E) Lateral view of wild-type and Pitx2 c^-/- 18.5 dpc embryos.