TY - JOUR
T1 - Human papillomavirus-16 E6/E7 transfected retinal cell line expresses the Muller cell phenotype
AU - Roque, Rouel S.
AU - Agarwal, Neeraj
AU - Wordinger, Robert J.
AU - Brun, Anne Marie
AU - Xue, Yaming
AU - Huang, Lan C.
AU - Nguyen, Loc P.
AU - Shay, Jerry W.
N1 - Funding Information:
This work was supported by NIH EY10766 and a Grant-in-Aid from the American Heart Association. * For correspondence at: Department of Anatomy and Cell Biology, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107–2699, U.S.A.
PY - 1997/4
Y1 - 1997/4
N2 - The introduction of viral transforming genes into mammalian cells has been used in establishing cultures of unlimited lifespan. Although Muller cells, the predominant glial cells in the mammalian retina, have been isolated using a variety of techniques, most of these cultures have limited capacity for cell division and are often contaminated by other cell types especially astrocytes, endothelial cells and microglial cells. We have established pure cultures of retinal cells which express Muller cell characteristics and exhibit unlimited growth in vitro. We now report the techniques involved in the propagation and characterization of these cultures. Mixed retinal cultures isolated from dystrophic rat retinas were infected with defective retroviruses coding for human papillomavirus (HPV) type 16 E6 and E7 proteins. The disabled viral constructs also contained the neomycin gene allowing selection of the cultures using Geneticin, a neomycin analogue. Pure cultures were then obtained from Geneticin-selected populations by limiting end-dilution techniques. The expression of the HPV- 16 E6/E7 genes in the transfected cell line was established using an HPV-16 E6/E7 PCR product to probe Northern blots. Cloned cells were found to be highly reactive for Muller cell markers including S-100, carbonic anhydrase- C, cellular retinaldehyde 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, cytoplasmic filaments, abundant perinuclear rough endoplasmic reticulum, and smooth endoplasmic reticulum extending to the cell processes. Growth patterns of late passage cells (> 50 passages) showed a lag phase of 48 hr followed by exponential growth extending past visual confluence at day 5. Since the cultures have undergone more than 240 population 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 Muller cells.
AB - The introduction of viral transforming genes into mammalian cells has been used in establishing cultures of unlimited lifespan. Although Muller cells, the predominant glial cells in the mammalian retina, have been isolated using a variety of techniques, most of these cultures have limited capacity for cell division and are often contaminated by other cell types especially astrocytes, endothelial cells and microglial cells. We have established pure cultures of retinal cells which express Muller cell characteristics and exhibit unlimited growth in vitro. We now report the techniques involved in the propagation and characterization of these cultures. Mixed retinal cultures isolated from dystrophic rat retinas were infected with defective retroviruses coding for human papillomavirus (HPV) type 16 E6 and E7 proteins. The disabled viral constructs also contained the neomycin gene allowing selection of the cultures using Geneticin, a neomycin analogue. Pure cultures were then obtained from Geneticin-selected populations by limiting end-dilution techniques. The expression of the HPV- 16 E6/E7 genes in the transfected cell line was established using an HPV-16 E6/E7 PCR product to probe Northern blots. Cloned cells were found to be highly reactive for Muller cell markers including S-100, carbonic anhydrase- C, cellular retinaldehyde 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, cytoplasmic filaments, abundant perinuclear rough endoplasmic reticulum, and smooth endoplasmic reticulum extending to the cell processes. Growth patterns of late passage cells (> 50 passages) showed a lag phase of 48 hr followed by exponential growth extending past visual confluence at day 5. Since the cultures have undergone more than 240 population 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 Muller cells.
KW - Carbonic anhydrase-C
KW - Cellular retinaldehyde binding protein
KW - Dystrophic retina
KW - Glial fibrillary acidic protein
KW - Glutamine synthetase
KW - Human papilloma virus-16 E6 and E7
KW - Immunoblotting
KW - Immunocytochemistry
KW - Microglial cells
KW - Northern blotting
KW - Retinal glia
KW - S-100
KW - Viral transfection
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U2 - 10.1006/exer.1996.0230
DO - 10.1006/exer.1996.0230
M3 - Article
C2 - 9227269
AN - SCOPUS:0031126950
SN - 0014-4835
VL - 64
SP - 519
EP - 527
JO - Experimental Eye Research
JF - Experimental Eye Research
IS - 4
ER -