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. 2005 Aug 15;19(16):1934–1950. doi: 10.1101/gad.1300705

Figure 1.

Figure 1.

Generation of keratin-5-matriptase transgenic mice. (A) Schematic structure of the keratin-5-matriptase transgene. The bovine keratin-5 promoter (K5), rabbit β-globin exons (E), rabbit β-globin intron B, mouse matriptase cDNA, rabbit β-globin polyadenylation signal (Poly A), and positions of primers used for mouse genotyping (a and b) and RT–PCR analysis (c and d) of transgene-specific matriptase mRNA are indicated. (B) Expression of the keratin-5-matriptase transgene in mouse skin. RT–PCR analysis of skin from wild type (lanes 1,3), littermate keratin-5-matriptase transgene-line-A (K5-Mat-A) (lane 2), and keratin-5-matriptase transgeneline-B (K5-Mat-B) (lane 4) using a transgene-specific primer pair as indicated in A, or a primer pair amplifying GAPDH mRNA. The positions of DNA fragments amplified from the transgene-derived matriptase mRNA (188 bp), and GAPDH (236 bp) are indicated with arrows. (C) Northern blot analysis of total matriptase mRNA in the skin of newborn wild-type and keratin-5-matriptase transgenic mice. The ratio of matriptase to GAPDH signal intensity is shown. (D) Quantitative real-time PCR analysis of relative endogenous matriptase mRNA expression in wild-type newborn skin, adult skin (4 mo), and DMBA/PMA-induced tumors (left panel), and relative transgenic matriptase mRNA expression in K5-Mat-A (middle panel) and K5-Mat-B (right panel) transgenic mice. The matriptase expression levels were normalized to GAPDH mRNA expression. (E) In situ hybridization of matriptase mRNA expression in newborn skin of wild-type and littermate K5-Mat-A transgenic mice (left panels), newborn wild-type and littermate K5-Mat-B transgenic mice (middle panels), and adult hyperplastic skin (1 yr old) from a K5-Mat-B-transgenic mouse (right panel) using 35S-labeled antisense probes and sense probes as indicated. Identical results were obtained with two nonoverlapping pairs of antisense and sense probes. (F) Quantitative real-time PCR analysis of total matriptase mRNA expression in primary cultured keratinocytes from K5-Mat-B and littermate wild-type mice. The matriptase expression levels were normalized to expression of GAPDH mRNA. (G) Matriptase gelatin zymography of concentrated conditioned media from cultured keratinocytes from three newborn wild-type pups (lanes 1–3) and three littermate K5-matriptase pups (lanes 4–6). The bracket indicates zones of gelatin lysis. (H) Optical scanning of the clarity of the 90-kDa lysis zones in G.(I) Coomassie-stained gel containing the samples used for matriptase gelatin zymography in G analyzed in parallel by SDS-PAGE to verify equal protein loading. (G,I) Molecular weight markers (in kilodaltons) are indicated on the left. (Arrowheads) Follicles; (arrows) interfollicular epidermis; N indicates the number of mice analyzed; error bars indicate standard error of the mean. Scale bars, 100 μm.