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D.C. Otteson, R. Lewis, D.J. Zack; Isolation of a Conditionally Immortalized Muller Cell Line . Invest. Ophthalmol. Vis. Sci. 2006;47(13):132.
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The purpose of these studies is to generate and characterize conditionally immortalized retinal cell lines using the Immortomouse, a transgenic mouse that carries a temperature–sensitive, SV40 large T–antigen that is inducible by IFN–gamma (IFN–γ).
Retinas from heterozygous Immortomouse (Charles River Laboratories) were dissociated in Papain and cultured in Neurobasal Medium containing B27, 20mM glutamine, 2% fetal bovine serum, penicillin/streptomycin and supplemented with IFN–γ. Immuno–staining of 4% paraformaldehyde fixed cells used standard methods. T–antigen expression was analyzed in cell lysates by western blot using antibodies against the SV40 large T–antigen and enhanced chemiluminescence.
Dissociated retinal cells from P10 mice were selected by their ability to adhere to poly–L–lysine/laminin coated dishes. Cultures of adherent cells were grown at 33C in the presence of 50 U/ml IFN–γ and have been maintained under these conditions for17 passages. Proliferation was evidenced by increases in cell number, the presence of mitotic figures and BrdU incorporation. After 4 passages, cultures contained large, adherent cells with multiple, irregular lamella–podia and large nuclei. All cells were strongly immuno–positive for vimentin, weakly positive for nestin and GFAP. No Pax2, Pax6 or PKC–alpha positive cells were present. Injury of monolayers in a scratch test resulted in increased GFAP immuno–staining in cells at the edge of the wound. By immuno–histochemistry, T–antigen expression was detectible in cells cultured with 50 or 100 U/ml IFN–γ, but not 0 or 25 U/m. By Western blot analysis, T–antigen was detected all concentrations of IFN–γ tested (10 to 200 U/ml).
We have isolated a conditionally immortalized cell line from the retinas of Immortomouse transgenic mice. Based on morphology, vimentin expression and low levels of GFAP expression, we have identified these as Muller glia. Future studies use gene expression profiling to examine differences between cells cultured under inducing (IFN–g; 33C), non–inducing (without IFN–g; 39C) conditions and acutely isolated Müller cells and to evaluate effects of continued passages in vitro.
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