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Fig. 1. Gene targeting at the Dkk2 locus and the cornea phenotype of Dkk2^–/– mutant mice. (A) Partial restriction maps of the wild-type Dkk2 locus, the targeting vector and the disrupted Dkk2 allele. Exon 1 and part of the intron 1 of the Dkk2 allele were replaced with the neomycin (neo) cassette. The expected size of EcoRI-generated fragments are 7 and 5.2 kb. RI, EcoRI; RV, EcoRV; H3, HindIII; Ap, ApaI. (B) Southern blot analysis: EcoRI-digested genomic DNA isolated from wild-type and three targeted ES cell lines (lanes 1-3) generated 7 kb and 5.2 kb bands, respectively, when detected with a 5' external genomic probe. ApaI-digested genomic DNA generated 7 kb and 5 kb bands for wild-type and mutant alleles, respectively, when detected with a 3' external genomic probe. (C,D) Corneal phenotype. An opaque plaque and hairs (arrow) are visible on the ocular surface of a Dkk2^–/– mutant (D), while an age-matched wild-type control eye contains a transparent cornea (C). (E-G) Paraffin sections of eyes stained with Hematoxylin/Eosin, indicating epithelial layer (ep) and the stroma (st). (E) Wild type. Corneal epithelial hyperplasia is evident in the Dkk2^–/– eye (F). (G) Higher magnification view of F; skin appendages such as hair follicles (arrow) and sebaceous glands (arrowhead) are indicated. (H) Periodic acid-Schiff base staining of the Dkk2^–/– cornea demonstrates presence of goblet cells (arrow). (I) Ectopic pigment-granules are present in the epithelial basal cells of the mutant cornea. The border between the epithelial (ep) and stromal (st) layers of a mutant cornea is indicated (broken line). Scale bars: in C, 1 mm for C,D; in E, 200 µm for E,F; 50 µm in H; 35 µm in I.

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