SUPPLEMENTARY INFORMATION

Figure S1 (pdf 17K)

Bacterial homologous recombination strategies used in the construction of Krt75 targeting vector. (A) Schematic representation of the wild type mouse Krt75 genomic locus in a BAC vector. Exons are represented by solid boxes. A selection marker - counter selection marker (SM-CSM), RpsL-neo, flanked with short DNA sequences derived from the N159 region in exon 1 of Krt75 (homologous arms) are shown. In the E. coli host, homologous recombination between these arms, indicated by , enabled the cassette to be inserted in exon 1. (B) Schematic representation of the mouse Krt75 genomic locus, after partial replacement of exon 1 with the RpsL-neo cassette. This BAC carries the neo-resistant gene, therefore, is resistant to kanamycin. Homologous recombination with a 362 bp PCR product of mouse Krt75, harboring the deletion of codon 159 (asterisk), then replaced the RpsL-neo cassette. Counter-selection for the absence of RpsL gene, which renders the E. coli host susceptible to streptomycin, allowed the growth of clones that contained the mutation. (C) Schematic representation of the mouse Krt75 genomic locus with the N159del mutation (asterisk) in a BAC vector. A neo-cassette, flanked by loxP sites and homologous regions to intron 1 of Krt75 is also shown. Homologous recombination through these regions, indicated by , enabled this cassette to be inserted in intron 1. Successfully inserted clones were resistant to kanamycin. (D) Subcloning of the modified Krt75 locus into a gene targeting vector. The modified mouse Krt75 locus and the neo-cassette inserted in intron 1 were subcloned by homologous recombination into a pUC-TK plasmid vector. Homologous recombination between the plasmid and BAC, as indicated by , generated the gene targeting vector. Asterisk (*) represents the N159del mutation. Representive only, not drawn to scale.

Figure S2 (pdf 22K)

Strategy to generate a mouse model expressing the Krt75 N159del mutation (equivalent to the human KRT6A N172del mutation). (A) Schematic representation of the wild type mouse Krt75 genomic locus. Exons are represented by solid boxes. The locations of codon 159 (159N) and probes for Southern blotting are shown. (B) Targeting vector. A 7.9 kb genomic sequence (including exons 1–5) of Krt75 was cloned in the pUC-TK vector. Deletion of codon 159 is indicated as an asterisk (*). A 3.2 kb neo-cassette, flanked by loxP sites, was introduced in intron 1. Homologous recombination between the targeting vector and the Krt75 gene locus in embryonic stem (ES) cells is symbolized by . (C) Predicted structure of the recombinant Krt75 gene locus in ES cells. Introduced restriction sites (Mfe I) in the neo-cassette in intron 1 are used to distinguish between the recombinant Krt75 locus and the wild type locus by Southern blotting. (D) Predicted structure of the recombinant Krt75 locus after Cre-mediated recombination. Cre-mediated recombination excises the neo-cassette from intron 1 before ES cells were injected into embryos. Representive only, not drawn to scale.

Figure S3 (pdf 142K)

Changes in vibrissae. (A, B) Normal wild type mouse vibrissa are relatively straight with a long slightly tapered shaft. Boxed area is enlarged (B). (C–E) Krt75tm1Der/Krt75tm1Der mutant mice had irregularly spaced bulges with clumping of pigment in the vibrissa. Boxed areas are enlarged (D, E). Scale bars=500 m in (A) and (C); 50 m in (B), (D) and (E).

Table S1 (pdf 47K)

Primers used in this study