(A) Collagen VII (ColVII) mRNA expression was analyzed in immortalized keratinocytes of Col7a1^WT/WT (lanes 1), Col7a1^flNeo/WT (lanes 2), and Col7a1^flNeo/flNeo (lanes 3) mice by RT-PCR. Amplification of exon 1–exon 2 showed no difference in expression levels between the genotypes. In contrast, amplification of exon 2–exon 3 revealed aberrant splice variants in Col7a1^flNeo/flNeo keratinocytes. Asterisks indicate the normal splice product (***) and the longest aberrant product (**). A transcript variant containing part of the PGK-Neo sequence (*) was identified in Col7a1^flNeo/flNeo and Col7a1^flNeo/WT cells. (B) Schematic representation of the major transcript variants, the majority of which cannot be translated into protein. The asterisks denote the variants shown in A. Black boxes indicate Col7a1 exons; gray boxes indicate the Neo cassette; bold black arrows indicate the promoter PGK; “Stop” indicates the translational stop of neomycin phosphotransferase; triangles indicate loxP sites; ovals indicate Frt sites. (C) Collagen VII protein levels were analyzed by immunoblotting extracts of dermis, immortalized keratinocytes, and fibroblasts of newborn Col7a1^WT/WT (lanes 1), Col7a1^flNeo/WT (lanes 2), and Col7a1^flNeo/flNeo (lanes 3) mice. Each individual lane contains samples of an independent animal. Collagen VII was detected with the NC2-10 antibody (44). As loading controls, the Coomassie blue–stained band of α1 chain of collagen I (ColI) or immunoblotting with anti–β-tubulin were used. Dermis and cell extracts of Col7a1^flNeo/flNeo mice contained strongly reduced amounts of collagen VII.