HUMAN PAPILLOMAVIRUS-16 E6 E7 TRANSFECTED RETINAL CELL-LINE EXPRESSESTHE MULLER CELL PHENOTYPE/

The introduction of viral transforming genes into mammalian cells has been used in establishing cultures of unlimited lifespan. Although Mul ler cells, the predominant glial cells in the mammalian retina, have b een isolated using a variety of techniques, most of these cultures hav e limited capacity for cell division and are often contaminated by oth er cell types especially astrocytes, endothelial cells and microglial cells. We have established pure cultures of retinal cells which expres s Muller cell characteristics and exhibit unlimited growth in vitro. W e now report the techniques involved in the propagation and characteri zation of these cultures. Mixed retinal cultures isolated from dystrop hic rat retinas were infected with defective retroviruses coding for h uman papillomavirus (HPV) type 16 E6 and E7 proteins. The disabled vir al constructs also contained the neomycin gene allowing selection of t he cultures using Geneticin, a neomycin analogue. Pure cultures were t hen obtained from Geneticin-selected populations by limiting end-dilut ion techniques. The expression of the HPV-16 E6/E7 genes in the transf ected cell line was established using an HPV-16 E6/E7 PCR product to p robe Northern blots. Cloned cells were found to be highly reactive for Muller cell markers including S-100, carbonic anhydrase-C, cellular r etinaldehyde binding protein, and glial fibrillary acidic protein but not for glutamine synthetase. Ultrastructural studies showed stacks of cells with long elaborate processes, short microvilli, coated pits, c ytoplasmic filaments, abundant perinuclear rough endoplasmic reticulum , and smooth endoplasmic reticulum extending to the cell processes. Gr owth patterns of late passage cells (> 50 passages) showed a lag phase of 48 hr followed by exponential growth extending past visual conflue nce at day 5. Since the cultures have undergone more than 240 populati on doublings, they can be characterized as a continuous cell line with unlimited lifespan. The HPV-16 E6/E7 transfected Muller cell line may prove useful in studies requiring abundant and pure cultures of Mulle r cells. (C) 1997 Academic Press Limited.