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Fig. 1. Transgene expression in corneal epithelia. (A) The ALDH3 promoter/Cdk5 construct, including the untranslated first exon and the translation start site in exon 2 (arrow). (B) Protein extracts were prepared from corneal epithelia isolated from wild-type (wt) and heterozygous ALDH3-Cdk5 transgenic (tg) littermates. Cdk5 expression was examined by immunoblotting using an anti-Cdk5-C-terminus-specific antibody. The lower panel shows Ponceau staining of protein in the same samples as a loading control. The experiment was performed in triplicate and images were quantified by densitometric scanning. The results indicated that Cdk5 expression in transgenic animals was elevated 3.0 times. (C) Immunofluorescence of Cdk5 in corneas of an adult normal mouse. Immunostaining was seen in all layers of the corneal epithelium (ep) and was most intense along the basal aspect of the basal cells (small arrows) in both normal and transgenic animals. In superficial cells, staining appeared to be associated with cell membranes (arrowhead). Keratocytes of the corneal stroma (s) were not significantly stained. (D) Immunofluorescence of Cdk5 in corneas of ALDH3-Cdk5 transgenic animals. Cdk5 accumulation was seen in apical region of basal and wing cells of the transgenic corneas (double arrow) as well as along the basal aspect of the basal cells (small arrows) and along membranes of the superficial cells (arrowhead). Bar, 100 µm (C,D). (E) Effect of transgene expression on corneal wound healing. A 1.5 mm debridement wound was made in the central cornea of age-matched wild-type and ALDH3-Cdk5 transgenic mice, at age 8-10 weeks. Initial wound areas were determined by image analysis of six eyes (four normal and two transgenic) immediately after wounding. Wound areas at 12 hours were determined by image analysis of 34 wild-type and ten transgenic eyes. The healed area was calculated by subtracting the average remaining wound area at 12 hours from the average initial wound area and was then expressed as a proportion of the average initial wound area. ALDH3-Cdk5 mice showed a significantly lower rate of wound healing than the wild type (P<0.01).
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