Based on the above references and our current findings, we have concluded that the RGC-5 cells are, indeed, 661W cells. This is supported by the following data: The RGC-5 cell line was of mouse and not rat cells origin, and RGC-5 cells appear to be genetically identical to 661W cells, and possess the same transforming protein, SV40 large T-antigen. How this happened still is a mystery. The 661W cell line was in use in the laboratory of the RGC-5 cell origin and when it was attempted to develop a transformed RGC line, may have led to 661W cell contamination during the cell cloning experiments. Why was this possibility not caught? At the time the clone was selected, there were several important characterization procedures that were not done. Why? Let's examine the procedure that was used and see where this could have been improved. Retinas obtained from postnatal day 1 rats were dissociated with papain and the mixed retinal cell population was seeded on 100 mm tissue culture dishes. After 4 hours in culture, the retinal cells were overlaid with a Ψ2E1A viral suspension and allowed to incubate for 4 hours for viral transduction to occur. The cells then were washed free of the viral suspension and stably transduced cells were selected using Geneticin (a neomycin analog). The surviving cells were presumed to be transformed by the virus (since they were neomycin resistant) and were used to select various clones of putative retinal ganglion cell lines. The selected clones arising from well separated single cells were selected by placing small circles of Whatman paper dipped in trypsin, lifting them off the dishes, and propagating them in 6-well plates. The clones propagated in this manner were used to test for markers of retinal ganglion cells. A major fault in this process was that the investigators made no attempt to determine if the transformed cells expressed the Ψ2E1A protein, which was instrumental to the cellular transformation. As seen in the present report, these “RGC-5” cells do not have the Ψ2E1A viral protein, but instead express the SV40 large T-antigen protein. According to the lead investigator who established the RGC-5 cell line, the cells from the clone were neomycin-resistant, and had a growing rate that suggested successful transduction and transformation. Moreover, the RGC-5 cells expressed retinal ganglion cell markers, Thy 1.1., Brn 3B or 3c, and related growth factors.These characteristics also were upheld by others in the field.In addition, following treatment with various agents that appeared to differentiate these cells morphologically, they resembled retinal ganglion cells.Some of these treatments resulted in glutamate-sensitive NMDA receptors expressed that responded to glutamate with increased calcium responses and increased cell death following increased dosing with glutamate.Early mistakes with insufficient characterization were not apparent because of the data of others suggested that these cells were, indeed, retinal ganglion cells. Recently Nieto et al. demonstrated that RGC-5 cells contained opsins and intrinsic light responses that were indicative of cone-like photoreceptors.The 661W cell line developed by Tan et al. is a mouse photoreceptor-derived cell line that was immortalized with the SV40 T-antigen and express cone proteins, but not those of rod photoreceptors.The finding that RGC-5 cells are more like cone photoreceptors agrees with our contention that the RGC-5 cell line is, indeed, the 661W cell line. Recently, Wood et al. have shown that visible light exposure on cultured RGC-5 cells can activate cell death pathways leading to apoptosis.The finding that “RGC-5” cells are susceptible to light is consistent with these cells being more like photoreceptor cells than ganglion cells.