Characterization of immortalized rabbit corneal epithelial cells with SV40 large T antigen

The authors developed a line of immortalized rabbit corneal epithelial cells by transfecting a primary culture of New Zealand White (NZW) rabbit corneal epithelial cells with Simian virus 40 (SV40) T antigen gene using a calcium phosphate precipitation method. The transfected cells, which survived more than 40 passages and 400 population doublings, showed some ability to form colonies in soft agar, and had doubling times and plating efficiencies not significantly different from those of untransfected cells. The epithelial nature of the transfected cells was confirmed by immunofluorescence staining with the antibody to 64 kD keratin (AE5), a specific marker for corneal type epithelial cells. Immunofluorescence microscopic examination revealed that the transfected cells expressed major basement membrane components of epithelial cells, including collagen types IV and VII and laminin. Electron microscopic studies demonstrated the presence of microvilli and intercellular in cultured transfected cells. These results indicate that the transfected cells retain some of the normal phenotypic characteristics of corneal epithelial cells and may therefore be used to study the corneal epithelium in vitro.