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. If has become apparent that one of the main difficul ties of this technology is the overall poor take of the grafts as a consequ ence of severely damaged dermal beds. Skin substitutes providing both cultu red keratinocytes, as an epidermal layer, and a dermal analogous offer a mo re suitable material for skin repair. Ex vivo transfer of stroma regenerati on-promoting genes to keratinocytes appears to be an attractive strategy fo r improving the therapeutic action of these grafts. The use of epidermal-sp ecific promoters as expression drivers of exogenous genes results in both h igh expression levels and stratum specificity as shown in transgenic mice s tudies. Most current gene transfer protocols to primary keratinocytes invol ve transduction of epidermal cells with retroviral vectors. However, transf er 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 endothelia l cell mitogen driven by the 5.2 kb bovine keratin K5 promoter, was stably transfected into pig primary keratinocytes. Genetically modified keratinocy tes, expanded on live fibroblast-containing fibrin gels and transplanted to nude mice as a composite material, elicited a strong angiogenic response i n the host stroma as determined by fresh tissue examination end CD31 immuno staining. Since the formation of a well-vascularized wound bed is a crucial step for permanent wound closure, the use of an 'angiogenic' composite mat erial may improve wound bed preparation and coverage with cultured keratino cyte grafts.