FIG. 2.

(a) The β-gal-Neo cassette was inserted into the second exon of the mouse gene for EPPK to generate the targeting vector used for generation of EPPK^−/− mice. As a result of homologous recombination in mouse ES cells, this construct replaced the second exon of the wild-type gene. The “outside” probe was generated by cleavage at the KpnI and XbaI sites for selection of ES cells and mice by genomic Southern blotting. K, KpnI; H, HindIII; Xb, XbaI; Xh, XhoI. (b) Southern blotting yielded a 19-kbp DNA fragment in the case of the wild-type allele and a 15-kbp fragment in the case of the mutant allele. Heterozygous animals harbored both alleles. (c) Wild-type and heterozygous skin cells expressed EPPK mRNA, whereas EPPK^−/− cells did not express this mRNA. All cells expressed β-actin mRNA.

(d) Histology of skin of the feet. Staining with hematoxylin and eosin revealed the absence of blistering in both wild-type and EPPK^−/− mice (top). Immunostaining with polyclonal antibodies against the linker region of EPPK was negative in EPPK^−/− skin, while it was positive in wild-type skin (bottom). Bars = 50 μm.