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 beta-actin (pbetaAct) 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 microM) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of pbetaAct.EGFP mice contained 270 microM EGFP, while their bipolars had 440 microM. The cones of pLMCOps.EGFP mice expressed 60 microM 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 pbetaAct.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 microM 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.
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 beta-actin (pbetaAct) 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 microM) were in the retinal pigment epithelium (RPE) cells of the AAV-transduced eyes. Living photoreceptors of pbetaAct.EGFP mice contained 270 microM EGFP, while their bipolars had 440 microM. The cones of pLMCOps.EGFP mice expressed 60 microM 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 pbetaAct.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 microM 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.
Authors: David H Lovett; Rajeev Mahimkar; Robert L Raffai; Leslie Cape; Bo-Qing Zhu; Zhu-Qiu Jin; Anthony J Baker; Joel S Karliner Journal: PLoS One Date: 2013-07-16 Impact factor: 3.240