Targeted expression of transgenic PIGF-2 in epidermal keratinocytes.

(A) Schematic representation of the K14–PlGF-2 transgene construct. A 533-bp human PlGF-2 cDNA fragment was ligated to the BamHI restriction site of the keratin 14 promoter cassette. (B) Overexpression of PlGF-2 mRNA in the dorsal skin of 3-week-old K14–PlGF-2 transgenic mice and in cultured epidermal keratinocytes isolated from PIGF-2 transgenic mice was confirmed by Northern blot analysis. Hybridization with a murine β-actin probe served as control for equal loading. (C) Western blot analysis of skin lysates demonstrates the presence of the intact PlGF-2 protein in PlGF-2 transgenic (TG) skin. Low levels of endogenous PlGF-2 were detected in wild-type (WT) skin. Conditioned media obtained from human dermal microvascular endothelial cells (HDMEC) served as positive controls. (D-E) Targeted overexpression of the K14–PlGF-2 transgene in the basal epidermal keratinocyte layer and in outer root sheath keratinocytes of hair follicles was confirmed by in situ hybridization in 2-week-old transgenic mice. (F-G) Low-level epidermal expression of the K14–PlGF-2 transgene was observed by in situ hybridization in adult, 8-week-old transgenic mice. Bright-field (D,F) and dark-field (E,G) micrographs. Scale bars = 50 μm.