The introduction of viral transforming genes into mammalian cells has been used in establishing cultures of unlimited lifespan. Although Müller cells have been isolated from mammalian retinas using various techniques, most cultures have limited capacity for cell division and are often contaminated by other retinal cell types especially microglial cells. We have established pure cultures of rat Müller cells using defective retroviruses coding for human papilloma virus (HPV) type 16 E6 and E7 proteins and we now report of their characteristics. Methods. Mixed glial cultures isolated from rat retinas were transfected with the disabled viral constructs coding for E6 and E7 proteins and resistance to neomycin. Transfected cultures were selected using 300 μg/ml Geneticin, a neomycin analogue, and selected cell populations were cloned by limiting end-dilution. The expression of the E6/E7 genes in the clonal cell line was determined using a HPV 16 E6/E7 cDNA probe in Northern blots. Clonal cultures were further tested for expression of glia-specific cell markers, growth patterns, and mitogenic activity. Results. Transfected glial cells were found positive for Müller cell markers including glial fibrillary acidic protein, cellular retinaldehyde binding protein, carbonic anhydrase-C, and S-100. Ultrastructural studies showed "cell stacks" devoid of tight junctions, abundant perinuclear rough endoplasmic reticulum and free ribosomes, and cortical layers of smooth endoplasmic reticulum extending to the long cellular processes. Growth patterns showed exponential growth even after visual confluence. Proliferation assays indicated the presence of endothelial cell and photoreceptor cell mitogens, as observed in primary Müller cell cultures. Conclusions. The Müller cell E6/E7 cell line should prove useful in studies requiring pure and abundant cultures of Müller cells which maintain their phenotype and retain their growth promoting activity.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Feb 15 1996|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience