Constitutive deletion of KtyII^−/− keratin gene locus. (A) Schematic representation of the keratin type II cluster. Green arrowheads identify type II keratin genes oriented in the direction of the tip. The pink arrowhead identifies the only type I keratin (Krt18) at the end of type II cluster. (B) 5′-targeting vector (MHPN117k13; Adams et al., 2004). (C) 3′-targeting vector (MHPP322c09; Adams et al., 2004). Gaps [GR] are introduced into the region of homology before targeting. (D) During homologous recombination, the gap is repaired. PCR primers to identify homologous recombinants spanned the gap and the proximal vector sequences (Table S1). (E) Cre-mediated recombination between loxP sites in cis leading to deletion of the keratin cluster. Southern probes are indicated as bold bars in B–E. (F) Southern blot analysis of ES cell clones targeted at the 5′ (i) and the 3′ end (ii) with a probe that distinguishes BsrGI (B) and HincII (Hi) fragments in the WT and targeted allele, respectively. (G) Southern blot analysis of ES cells after Cre-mediated recombination using unique probes spanning the 5′ and 3′ hprt. Probes distinguish double-targeted 8.3-kb and recombined 4.9-kb HinDIII (H) fragments using the 5′ hprt probe (i) and double-targeted 14.3-kb and recombined 10.5-kb EcoRI (E) fragments using the 3′ hprt probe (ii). (H) mRNA expression of keratins and vimentin (Vim) in E9.5 WT and mutant embryos. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalization control. (I) Immunofluorescence of K8/ K18 desmoplakin (DP) on sections of E9.5 WT and KO embryos. Bars, 10 µm.