A forensic path to RGC-5 cell line identification: Lessons learned

In 2001, a transformed cell line RGC-5 was developed from the rat retina that was thought to be of retinal ganglion cell origin. Since that time many investigators have used this line in a wide variety of studies to understand better retinal ganglion cell activity, cell signaling, and neuroprotection. Recently, a publication emerged that claimed that this RGC-5 cell line was derived from mouse and not rat, and other studies also indicated the expression of certain proteins that typically were not associated with retinal ganglion cells. This certainly came as a shock not only to the originators of this cell line, but also to others who have been using this as an in vitro model of rat retinal ganglion cells. As a result, we undertook experiments to determine if the RGC-5 cell line currently in use may have been mischaracterized. We, indeed, found that the RGC-5 cell line was of mouse and not rat origin, as was claimed originally in the original research report. We further determined whether these cells were of retinal ganglion origin. Our findings showed conclusively that RGC-5 cells were, indeed, of mouse origin and, using additional cytogenetic profile testing, karyotyping, and genetic and protein profiling, we concluded that these cells were not of retinal ganglion cell origin, but were the cell line 661W, a mouse SV-40 T antigen transformed photoreceptor cell line. The 661W cell line also was present in the laboratory of the originating laboratory and probably resulted in crosscontamination. The present study reviews some of the errors that were made in misidentifying the RGC-5 cell line and offers some insight as to how this may have happened, and ways one can avoid mischaracterization of a potentially important cell line.