Quantification of EGFP Expression in Living Cells of the Mouse Retina and Parallel Electrophysiological Assessment of Cell Health

Abstract

Abstract: : Purpose:To quantify and assess the effect of enhanced green fluorescent protein (EGFP) expression in live mouse retinal cells. Methods:Retinas of euthanized mice were isolated, and slices maintained in physiological saline or fixed for cryosectioning. The fluorescence of EGFP was measured with a photon–counting confocal laser scanning microscope¹. EGFP concentration in individual cells was determined with customized software. Expression levels were compared in virally infected mice (AAV2/5.CMV.EGFP or AAV2/2.CMV.EGFP), and mice expressing EGFP under the ß–actin (pßAct) or human L–cone opsin (pHLCOps) promoters. ERGs and suction pipette recordings of single rods were used to assess retinal function. Results:Control experiments revealed that the EGFP fluorescence level was reduced 5–fold or more of the fluorescence upon tissue fixation. Data collected from fixed sections were corrected for this effect. The highest observed level of expression, 1.4 mM, was in the cytoplasmic spaces of AAV2/5 transduced RPE cells. Peak expression in live rods (pßAct) averaged 294 µM; these mice had normal ERGs and recordings of individual rods were completely normal. EGFP expression was higher with AAV2/5 transduction than with AAV2/2. But, transduction with either virus was higher than that with pßA– or pHLCOps in the same cell type. Potential reasons for these differences in expression include target cell specificity, integration site, copy number, and promoter activity. Conclusions: (1) It is feasible to quantify and compare transgenic EGFP expression levels in individual cells of the live mouse retina. (2) Rods function normally with nearly mM levels of EGFP, and other retinal cells types appear to function normally with even higher levels. This allows the assessment of promoter efficacy and methods of delivery of genes to retinal cells. Footnotes ¹ J. Peet et al., J. Cell Science, in press.

Keywords: retina • microscopy: confocal/tunneling • gene transfer/gene therapy