FGF22 in Skin Development and Repair Figure 5. DMBA-induced tumorigenesis study. Cohorts of female mice (n = 10) were subjected to classical two-step skin carcinogenesis treatment. A) At 58 weeks post initiation, when the experiment was terminated, 100% of DMBA treated wild type mice had developed papillomas while only 50% of knockout treated mice had papillomas. Control TPA treated mice of both genotypes never developed any neoplastic skin lesions. B) Average number of papillomas per mouse at the indicated times after the start of DMBA/TPA treatment. With the exception of one outlier knockout female that developed five papillomas, the remaining knockout mice never developed more than one papilloma per mouse. The maximum number of papillomas on a DMBA treated wild type female was two. In addition to regression of some papillomas over time, when mice died, the average papilloma count was re-calculated for the remaining live mice, thus the average count could go up and down over the course of the experiment. Circles represent mice killed due to papillomas reaching the maximum size specified by Home Office licence. Asterisks represent deaths due to unrelated reasons. C) H&E staining of 4 mm sections from papillomas of wild type and fgf22 knockout mice showed identical histology, with exophytic growth and no sign of epithelial invasion (CS cornified squames; Ep epithelium; S stroma). D) Cartoon illustrating difference in response of wild type, fgf22 knockout and skin-specific fgfr2b knockout mice to chemically induced carcinogenesis and a proposed model for the phenotypes seen. We hypothesise that lack of downstream cytoprotective signalling mediated via Fgfr2b could potentially promote a more pro-tumorigenic phenotype. doi:10.1371/journal.pone.0039436.g005 Materials and Methods Generation of fgf22 Knockout Mice A gene targeting construct was made by flanking a Neo selection marker cassette with fragments of DNA homologous to the fgf22 genomic locus. The 59 arm consisted of 5 kb of genomic sequence 59 to the translation start site of fgf22, in exon 1, and the 39 arm comprised 1.2 kb sequence 39 to exon 3 of fgf22. 129ola mouse embryonic stem cells were used as a genomic DNA source. The targeting vector, constructed in pBluescript SK+ (Stratagene, La Jolla, Ca, USA), thus was designed to eliminate the entire coding sequence of fgf22 by deleting the coding region of exon 1 and the whole of exons 2 and 3. The targeting vector DNA was transfected into isogenic 129ola ES cells and G418 resistant clones were screened by Southern blot for correct homologous recombination. Of a total of 192 ES clones analysed, 2 correctly targeted clones displayed identical hybridisation patterns and were injected separately into C57Bl6/J blastocysts. The resulting chimeras were mated with wildtype C57/BL6 female mice to obtain germline transmission. Southern Blotting Genomic ES cell DNA was digested overnight with EcoRI and separated by electrophoresis on a 0.8% Agarose/TBE gel. The gel was photographed under UV illumination and then DNA was transferred overnight in 0.4 M NaOH onto a positively charged nylon membrane (Nytran SuPerCharge, Whatman, Maidstone, UK). A P32-dCTP-labelled screening probe (59Fgf22), correspond- ing to 400 bp of fgf22 genomic sequence upstream of the 59 homology arm, was prepared using Ready-To-Go DNA Labelling Beads (GE Healthcare, Amersham, UK) according to the manufacturer’s instructions. The membrane was hybridized with 50 ng of 32P-labelled probe, exposed overnight to a storage phosphor screen and read on a Storm 860 phosphorimage scanner (Molecular Dynamics, Sunnyvale, Ca, USA). PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39436