PURPSOE: To determine whether feline immunodeficiency virus (FIV) can transduce retinal ganglion cells (RGCs) in culture.
METHODS: The two step immunopanning technique was used to isolate the RGCs from post-natal day 8 Sprague Dawley rats. Purified RGCs were maintained in serum free medium with growth factors. The cells were then transduced at a titer of 3*10e7 or 3*10e5 with a novel minimal FIV vector containing the lacZ reporter gene under control of an internal CMV promoter (FIV2/CMV/lacZ). Two controls were used: (1) cells infected with a mock virus, and (2) cells not infected with virus. LacZ expression was detected by X-gal staining 24 and 48 hours after transduction in successfully transfected cells.
RESULTS: The expression increased with increasing incubation time and with increasing viral titer. LacZ expression 24 hours after transduction was found in 1.04% of cells transduced at a titer of 3*10e5 and 84.70% of cells transduced at a titer of 3*10e7. LacZ expression 48 hours after transduction increased to 2.21% in cells transduced at a titer of 3*10e5 and increased to 98.40% in cells transduced at a titer of 3*10e7. No X-gal staining was detected in the control cells.
CONCLUSIONS: Retinal ganglion cells in culture can be successfully transduced using a novel minimal FIV vector achieving a very high transduction efficiency. FIV mediated gene transfer to cultured RGCs may provide a good in vitro system for the determination of the effects of cell survival genes in delaying apoptotic RGC death.
Supported by the Glaucoma Research Foundation.