Fig. 5. Targeted disruption of the EP/AN enhancer. (A) Wild-type Otx2 allele, targeting vector and recombinant allele. The black box indicates the SpeI-BglII 559 bp region (AN) that is replaced with a neomycin-resistant gene (Neo, white boxes) flanked by loxP sequences (black triangles). DT-A is the diphtheria toxin-A fragment gene with MCI promoter, which is used for negative selection of homologous recombinants (Yagi et al., 1993b). Thick and thin lines indicate genomic and vector-derived sequences, respectively. Probe A is the Southern blotting probe used for identification of homologous recombinant ES cells displayed in the right panel. (B) Otx2 expression at E6.5 by whole-mount in situ hybridization (a,b) and by quantitative RT-PCR (c). +/+ and AN/AN denote wild-type embryos and homozygous mutants lacking the SpeI/BglII region, respectively. (C) Otx2^AN/– mutant phenotype at E6.5. Normal expression of cerberus-like in anterior visceral endoderm (a) and of T in primitive streak (b). Scale bars: 100 µm.