Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter.

Cultured epithelial grafts have proven to be life-saving in the treatment of large skin losses. It has become apparent that one of the main difficulties of this technology is the overall poor take of the grafts as a consequence of severely damaged dermal beds. Skin substitutes providing both cultured keratinocytes, as an epidermal layer, and a dermal analogous offer a more suitable material for skin repair. Ex vivo transfer of stroma regeneration-promoting genes to keratinocytes appears to be an attractive strategy for improving the therapeutic action of these grafts. The use of epidermal-specific promoters as expression drivers of exogenous genes results in both high expression levels and stratum specificity, as shown in transgenic mice studies. Most current gene transfer protocols to primary keratinocytes involve transduction of epidermal cells with retroviral vectors. However, transfer of gene constructs harboring these long DNA fragment promoters cannot be achieved through viral transduction. In this paper, we describe a protocol consisting of lipid-mediated transfection, G418 selection and an enhanced green fluorescence protein (EGFP)-based enrichment step for obtaining high levels of transgene-expressing primary keratinocytes. Using this protocol, the cDNA for vascular endothelial growth factor (VEGF), a potent endothelial cell mitogen driven by the 5.2 kb bovine keratin K5 promoter, was stably transfected into pig primary keratinocytes. Genetically modified keratinocytes, expanded on live fibroblast-containing fibrin gels and transplanted to nude mice as a composite material, elicited a strong angiogenic response in the host stroma as determined by fresh tissue examination and CD31 immunostaining. Since the formation of a well-vascularized wound bed is a crucial step for permanent wound closure, the use of an 'angiogenic' composite material may improve wound bed preparation and coverage with cultured keratinocyte grafts.