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D.C. Chung, Z. Wei, J. Bennett; In vivo Non–Viral Gene Transfer to Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):836.
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© ARVO (1962-2015); The Authors (2016-present)
To improve the efficiency of in vivo non–viral gene transfer in retinal ganglion cells.
Adult (3–4 month old) C57Bl/6 mice were anesthetized, and injected unilaterally with 1.5–2 ul of plasmid carrying CMV–driven enhanced Green Fluorescent Protein (eGFP) into the vitreous cavity. In additional cohorts of mice, eyes were injected with plasmid alone or with plasmid dissolved in mannitol to a final concentration of 20 % vol/vol. Additional control eyes received vehicle only or plasmid DNA without electroporation. Eyes were then electroporated with tweezer electrodes on a BTX ECM 830 electroporator (San Diego, CA). Electroporation parameters ranged from 5 to 80 volts (0.6 v/mm to 10 v/mm), 5 to 10 pulses and 50 to 100 millisecond pulse intervals. Final DNA concentration ranged from 1.39 to 10.0 mg/ml. Three days after injection/electroporation, animals were euthanized, eyes enucleated and fixed and retinal flat mounts were prepared. Areas containing eGFP were documented. Retinal flat mounts positive for eGFP expression were then cryoprotected, cross–sectioned at 10 um and evaluated for cellular specificity of eGFP. Numbers of eGFP–positive cells were counted.
EGFP–specific fluorescence was exhibited in retinal ganglion cell bodies and axons following intravitreal injection at a higher rate in animals receiving mannitol, compared to those with vector only. Control animals injected with buffer or with plasmid but not electroporated exhibited minimal fluorescence. Higher voltages and plasmid concentrations appeared to improve efficiency.
Increased efficiency of non–viral in vivo gene transfer into adult C57Bl6 mouse retina ganglion cells results from inclusion of mannitol in the plasmid solution.
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