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. 2014 May 27;289(28):19435–19447. doi: 10.1074/jbc.M114.553867

FIGURE 1.

FIGURE 1.

Generation and characterization of transgenes. A, schematic depiction of the transgenes used in this study. The human KRT14 gene promoter was used to drive the tissue-specific expression of keratin cDNAs (KRT8 and KRT8bc), and the rabbit β-globin intron (rβg Int) and human KRT14 3′-UTR sequence (Poly(A)) serve to stabilize the transgene mRNA in mouse cells. B, relative transgene copy number analysis was performed by conventional PCR of genomic DNA with transgene-specific primers. Krt16 was used as a single copy number reference gene. # indicates the highest relative copy number line for each transgene. C and D, quantitative RT-PCR analysis of transgenic human KRT8 mRNA (C) and endogenous mouse Krt14 mRNA (D) in back skin harvested from sex-matched 6-week-old animals. Relative RNA amount is normalized to both actin and Gapdh. CTRL indicates Krt5+/+ skin. Error bars represent S.E. A one-way analysis of variance (Dunnett's test) was used to test for significance, and the adjusted p values are reported. n.s., not significant; *, p < 0.04; **, p < 0.002. E, analysis of total skin protein extracts (10 μg/lane) by Western blotting in 6-week-old sex-matched adult animals. Two mice were analyzed for each of four transgenic lines; CTRL indicates Krt5+/+ skin. F, analysis of transgene expression in frozen skin sections of 6-week-old sex-matched adult animals. K8 epitopes are only present in basal layer keratinocytes (see brackets) of KRT8bc-1 and KRT8-1 transgenic epidermis and co-localizes with endogenous K14 in a normal keratin filament network. Control indicates Krt5+/+ skin. Arrows depict the interface between the epidermis (Epi) and dermis. Bars, 10 μm. wks, weeks.