The goal of this laboratory is to stably introduce and express foreign genes in keratinocytes of miniature swine (MS) epidermis. We have successfully used both an ex vivo approach and a direct in vivo approach to introduce genes into miniature swine epidermis. The inserted gene in these studies was the beta-galactosidase gene driven by strong viral promoters such as CMV, RSV, and SV40. In the first ex vivo approach, keratinocytes are isolated from MS epidermis; transfected with the beta-galactosidase gene which has been coated with liposomes or cationic lipids; and grafted back onto the donor pig as keratinocyte sheets. For this ex vivo approach, we have determined the optimal growing conditions of MS keratinocytes in tissue culture by analyzing different substrates on which to grow the cells, by testing different formulations of growth factors in culture media, and by optimizing the Ca+ concentration of the culture media. For transfection purposes, we have also optimized the lipid composition of the coating liposomes and the lipid to DNA ratio. Using these conditions, we have been able to transfect approximately 50% of plated keratinocytes. The second ex vivo approach is to transfect DNA into epidermal keratinocytes of a skin organ culture. This has also been successful and sometimes results in gene uptake and expression around hair follicles. We have re-grafted one of these organ cultures back onto the donor pig to see if expression of the transfected gene persists. We also have used two different in vivo approaches to directly introduce genes into MS epidermal keratinocytes. The first in vivo approach is to inject the DNA mixture directly into a skin blister where epidermis has been separated from underlying dermis. The goal is to transfect the rapidly proliferating basal keratinocytes which are re-epithelializing the dermal base of the blister. This approach has been successful in introducing genes into both the re-epithelializing keratinocytes as well as the keratinocytes in the epidermis of the blister roof. The second in vivo approach we have used is to inject the DNA mixture sub-epidermally (superficial dermis) with the resultant uptake and expression of our gene in keratinocytes of the overlying epidermis. To increase the number of keratinocytes containing and expressing our gene in vivo, we have begun developing methods to in vivo select for these transfected keratinocytes in a manner analogous to selection in tissue culture for stably integrated clonal cells using selecting agents such as the aminoglycoside antibiotics, G418(Geneticin) and hygromycin B. We have constructed plasmids containing both the beta-galactosidase gene and genes conferring resistance to the aminoglycoside antibiotics. The plasmids which contain the resistance genes will be used to select for keratinocytes which have been transfected with them. Pilot studies have shown that topical application of the aminoglycoside antibiotics to MS epidermis is capable killing normal keratinocytes, suggesting that in vivo selection is a feasible method. Additionally, this technique of topical epidermal selection can be applied and used in both the ex vivo and in vivo approaches described above.
