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Quantification of green fluorescent protein expression in mouse retinal ganglion cells following intravitreal injection of recombinant adeno‐associated virus
Author(s) -
Khatib T.,
Osborne A.,
Widdowson P.,
Martin K.
Publication year - 2016
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2016.0537
Subject(s) - green fluorescent protein , transduction (biophysics) , biology , titer , microbiology and biotechnology , recombinant dna , retinal , virus , viral vector , virology , gene , genetics , biochemistry
Purpose To determine the transduction efficiency and spatial pattern of green fluorescent protein (GFP) expression in mouse retinal ganglion cells (RGCs) following intravitreal injection of recombinant adeno‐associated virus (rAAV2). Methods 16 adult C57bL/6 mice received intravitreal injections of rAAV2‐CAG‐GFP at 3 different titres: 1 × 10E11, 1 × 10E12 or 1 × 10E13 genomic particles/ml. 2 μ l of the virus was injected at each concentration. To determine the effect of volume on transduction efficiency, a subset of animals received 1 μ l of the highest viral titre. Animals were sacrificed 21 days after injection. Retinal wholemounts were immunostained with Brn3a to identify RGCs and quantification carried out using imageJ software. The percentage of GFP positive RGCs was measured at each titre using Volocity software at 20× magnification. A further 20–25 images at 10× magnification were captured for each titre and merged using to reconstruct the entire wholemount to assess the spatial pattern of expression. Results RGC transduction rate increased with viral titre; 10% at 1 × 10E11, 53% and 1 × 10E12 and 64% at 1 × 10E13 genomic particles/ml. The volume injected did not appear to affect the transduction efficiency, with 64% of RGCs transduced at the highest titre using either 1 or 2 ul injection. GFP intensity also increased with viral titre with the spatial pattern of GFP expression more extensive at the highest titre. GFP expression at lower titres tended to localise around the injection site. Conclusions GFP transduction efficiency of RGCs can be quantified efficiently using Volocity software. We have demonstrated an increase in GFP expression and spread at higher viral titres with similar transduction efficiency at a lower volume.