Fig 1. Generation of Spink5 mutant mice.

(A) Nucleotide and amino acid sequences of human SPINK5/LEKTI (left). The two-bp deletion 398delTG in exon5 of human SPINK5 gene results in a frame-shift and PTC (red box) as described in Raghunath et al., 2004(23). Comparison of corresponding sequences in murine Spink5/LEKTI (right) where deletion of TG nucleotides at position 402 (black underline) has the same impact as in humans. (B) Position of critical TG nucleotides (black underline) in the exon5 of murine Spink5 gene. TALEN-binding sites are marked with red underline, position of primers for PCR screening (F1, R1, F2, R2) are denoted. As a targeting construct, a single-stranded oligonucleotide having sequences homologous to wt DNA (blue underline) flanked desired mutation. Premature STOP codon (red box) and introduced new XbaI recognition sequence are depicted. (C) RFLP analysis of targeted mice. PCR product amplified from genomic DNA using primers F1 and R1 was digested using XbaI enzyme. Cleavage products of 283 and 220 bp originate from the positively targeted allele, a 503 bp fragment marks the wt allele. (D) Expression of Spink5 mRNA was analysed by semi-qPCR analysis in Sp5^A135X/A135X mice using primers F2 and R2. Expression of GAPDH was used as a control. (E) Phenotype of Sp5^A135X/A135X newborn pups. Areas of peeling skin are marked with black arrowheads.