Fig. 2. Construction of the Brn3c-AP allele and genotype of Brn3b/Brn3c double knockout mice. (A) The 5' and 3' homology arms of Brn3c were inserted into the multiple cloning site of the AP-ki targeting vector (Gan et al., 1999). The targeting vector contains TK, neo and AP (hPLAP) cassettes as depicted. The position and exon/intron organization of the Brn3c gene with a 1.5 kb Sac1- BamH1 fragment used for genotyping are shown below the wild-type gene. B, BamHI; E, EcoRI; H, HindIII; S, SacI; V, EcoRV; X, XbaI. (B) Genotypes from a Brn3b^–/–:Brn3c (+/–) intercross using Southern hybridization. Genomic DNA was digested with HindIII and BamHI. Genotyping for Brn3b was performed as detailed by Gan et al. (Gan et al., 1999), and a band at 6-kb representing the targeted allele is observed in all lanes. Genotyping for Brn3c was performed using the 3' probe shown in A, which resulted in a wild-type band (7.1 kb) and a mutant band (5.5 kb). The three right hand lanes represent genotypes of Brn3b/Brn3c double mutant mice.