CHARACTERIZATION OF IMMORTALIZED RABBIT CORNEAL EPITHELIAL-CELLS WITHSV40 LARGE T-ANTIGEN

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