ESTABLISHMENT AND CHARACTERIZATION OF A RETINAL MULLER CELL-LINE

PURPOSE. Primary cultures of Muller cells have proven useful in cell b iologic, developmental, and electrophysiological studies of Muller cel ls. However, the limited lifetime of the primary cultures and contamin ation from non-neural cells have restricted the utility of these cultu res. The aim of this study was to obtain an immortalized cell Line tha t exhibits characteristics of Muller cells. METHODS. Primary Muller ce ll cultures were prepared from retinas of rats exposed to 2 weeks of c onstant light. Cells were immortalized by transfection with simian vir us 40. Single clones were obtained by repeatedly passaging cells using cloning wells. Immunocytochemical and immunoblotting studies were car ried out with glial fibrillary acidic protein (GFAP)specific and cellu lar retinaldehyde-binding protein (CRALBP)specific antibodies. Transie nt transfections with CRALBP-luciferase constructs were performed by e lectroporation. RESULTS. Oncogene transformation resulted in the estab lishment of a permanent cell line that could be readily propagated. Im munocytochemical and immunoblotting studies demonstrated that the Mull er cell line, nMC-1, expressed both GFAP, a marker far reactive gliosi s in Muller cells, and CRALBP, a marker for Muller cells in the adult retina. Transient transfection assays showed that promoter-proximal se quences of the CRALBP gene were able to stimulate reporter gene expres sion in rMC-1. CONCLUSIONS. Viral oncogene transformation has been suc cessfully used to isolate a permanent cell line that expresses Muller cell phenotype. The rMC-1 cells continue to express both induced and b asal markers found in primary Muller cell cultures as well as in the r etina. The availability of rMC-1 should facilitate gene expression stu dies in Muller cells and improve our understanding of Muller cell-neur on interactions.