Figure 1. AND-34 targeting. A: Exon structure of AND-34 cDNA is shown. B: Production of the recombinant ES clone is demonstrated. A genomic phage clone containing exons 4 and 5 was mapped as shown. The area between the indicated SmaI sites (3.5 kb) was deleted, and the two surrounding “arms”, 5.3 kb of the 5′ sequence and 1.8 kb of the 3′ sequence, were cloned into the pPNT vector. Location of wild type and knockout PCR primers are shown as well as the hybridization probe used for Southern blotting. C: Screening for homologous recombination. DNA prepared from ES clones was purified, aliquots from four clones per lane were pooled, and PCR was performed to detect the recombined KO allele. The first lane shows a pool that contains an ES clone that has the KO allele (arrows indicate lanes of PCRs with clones that contain no AND-34^−/− allele). The control PCR used primers specific for Neo. Homologous recombination was confirmed by Southern blot analysis as shown. DNA from individual clones was cleaved with BamHI and analyzed with the indicated probe. Clones 165 and 169 display the recombinant allele. Wild type 129 mouse DNA serves as a control. M=marker lane.