Purpose. To optimize parameters for efficient, cell-type specific retinal gene delivery, a small reporter gene detectible at low copy number is needed. We therefore adapted the jellyfish green fluorescent protein gene (GFPj) for expression in mammalian cells and tested it by Adeno-Associated Virus (AAV)-mediated delivery to the retina of experimental animals. Methods. A 717bp synthetic GFP gene containing 92 base substitutions altering 88 codons (GFPH1) was made, inserted into a recombinant AAV vector with a CMV promoter, packaged and tested in both tissue culture cells and guinea pig retina. A second gene version (GFPH2) containing the GHPH1 sequence with a Ser65Thr change was similarly tested. Results. Human 293 cells infected at low multiplicity (MOI of 1) were examined by fluorescence microscopy. 11% of the GFPH1 and 23% of the GFPH2 transduced cells showed gfp fluorescence. In contrast, only 0.5% of cells with GFPj were fluorescent. We injected the AAV-GFPH2 construct (20 μl of 109 infections particles per ml) into the posterior vitreous of guinea pigs. Fluorescence was seen in ganglion cells near the injection site and in a few horizontal cells. However, highest GFPH2 signal was in the adjacent RPE. Conclusion. Codon usage optimization within the GFP cDNA enhanced fluorescence detection at low copy number by approximately 22-fold with an additional 2-fold increase for the Ser65Thr substitution. Intravitreally injected of rAAV virus penetrates the entire retina from the vitreous to the RPE, infecting at least ganglion, horizontal and RPE cells enroute. To obtain retinal cell type-specific expression, neuronal and photoreceptor-specific promoter/GFPH2 constructs are being tested.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Feb 15 1996|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience