The distribution, concentration, and toxicity of enhanced green fluorescent protein in retinal cells after genomic or somatic (virus-mediated) gene transfer

Tonia S. Rex, John A. Peet, Enrico M. Surace, Peter D. Calvert, Sergei S. Nikonov, Arkady L. Lyubarsky, Elisabeth Bendo, Thomas Hughes, Edward N Pugh Jr, Jean Bennett

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

Purpose: The concentration of enhanced green fluorescent protein (EGFP) in individual photoreceptor cells of live mouse retina was quantified and correlated with physiological measurements of cell function. Methods: EGFP protein levels in the retinas of mice injected subretinally by either one of two serotypes of adeno-associated virus (AAV; AAV2/5.CMV.EGFP; AAV2/2.CMV.EGFP) were quantified with a photon-counting confocal laser scanning microscope and compared with those of transgenic mice whose retinas expressed EGFP under the β-actin (pβAct) or human L/M-cone opsin (pLMCOps) promoter. Single-cell suction pipette recordings of single rods and whole-field electroretinograms (ERGs) were performed to assess retinal cell function. Results: The highest levels of EGFP (680 μM) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of pβAct.EGFP mice contained 270 μM EGFP, while their bipolars had 440 μM. The cones of pLMCOps.EGFP mice expressed 60 μM protein. The amplitudes of the major components of ERGs were within the normal range for all transgenic animals examined, and single cell recordings from living pβAct.EGFP rods were indistinguishable from those of controls. Conclusions: EGFP levels in individual cells of live mouse retinas can be quantified, so that the efficacy of gene transfer methods can be quantified. Concentrations of several hundred μM are not deleterious to normal function of photoreceptors and bipolar cells. This approach can also be used to quantify levels of biologically active EGFP fusion proteins.

Original languageEnglish (US)
Pages (from-to)1236-1245
Number of pages10
JournalMolecular Vision
Volume11
StatePublished - Dec 30 2005
Externally publishedYes

ASJC Scopus subject areas

  • Ophthalmology

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    Rex, T. S., Peet, J. A., Surace, E. M., Calvert, P. D., Nikonov, S. S., Lyubarsky, A. L., Bendo, E., Hughes, T., Pugh Jr, E. N., & Bennett, J. (2005). The distribution, concentration, and toxicity of enhanced green fluorescent protein in retinal cells after genomic or somatic (virus-mediated) gene transfer. Molecular Vision, 11, 1236-1245.