Establishment of epidermal cell lines derived from the skin of the Atlantic bottlenose dolphin (Tursiops truncatus)

Abstract

The Atlantic bottlenose dolphin (Tursiops truncatus), a marine mammal found off the Atlantic coast, has become the focus of considerable attention because of an increasing number of mortality events witnessed in this species over the last several years along the southeastern United States. Assessment of the impact of environmental stressors on bottlenose dolphins (BND) has been difficult because of the protected status of these marine mammals. The studies presented herein focused on establishing epidermal cell cultures and cell lines as tools for the in vitro evaluation of environmental stressors on BND skin. Epidermal cell cultures were established from skin samples obtained from Atlantic BND and subjected to karyotype analysis. These cultures were further characterized using immunohistochemical methods demonstrating expression of cytokeratins. By two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we observed that the proteomic profile of BND skin tissue samples shared distinct similarities with that of skin-derived cultures. Epidermal cell cultures were transfected with a plasmid encoding the SV40 small t- and large T-antigens, as well as the neomycin-resistance gene. Five neomycin-resistant clones were isolated and expanded, and all of them proliferated at a faster rate than nontransfected BND epidermal cultures, which exhibited signs of senescence. Cell lysates prepared from two transfected clones were shown to express, by Western blot analysis, both SV40 tumor antigens. These experimental results are consistent with the concept that transfected clones expressing SV40 tumor antigens represent immortalized BND cell lines. Epidermal cell lines derived from Tursiops truncatus will provide a unique tool for studying key features of the interaction occurring between dolphins and the environment in which they live at their most crucial interface: the skin.