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D.S. Kuo, K.P. Greenberg, S.F. Geller, J.G. Flannery; In vivo Plasmid Tracking in Electroporated Rat Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5195.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Delivery of exogenous DNA to the mammalian retina by in vivo electroporation is proving to be a valuable tool for genomic, therapeutic, and physiological studies of vision. We sought to optimize experimental parameters to obtain the highest efficiency and specificity of delivery by using rhodamine–conjugated plasmids electroporated into the wild–type rat retina. Methods:Rhodamine dye was covalently attached to plasmid carrying CMV–driven Enhanced Green Fluorescent Protein (EGFP). For in vitro analysis, rhodamine labeled CMV–EGFP plasmid was transfected into 293T cells, which were fixed 24 hours later in 4% formaldehyde and visualized by fluorescence microscopy. Sprague–Dawley rat pups were anesthetized, injected (subretinally or intravitreally) with labeled plasmid, and electroporated. Control rats received identical injections and no subsequent electroporation treatment. Eyes were harvested 1 hour after injection and cryosectioned at a thickness of 12 µm. Sections were stained with Hoechst 33258 or DAPI to label cell nuclei and visualized with a fluorescence microscope. Results:Rhodamine and EGFP were both visualized in 293T cells, demonstrating that labeled plasmid was easily tracked and that the labeling process did not significantly affect plasmid expression. Electroporated eyes injected intravitreally showed plasmid localized to retinal ganglion cell nuclei after 1h, while animals treated subretinally showed plasmid localized in the subretinal space (SRS), retinal pigment epithelium (RPE), inner and outer segments, and outer nuclear layer. Control animals injected intravitreally showed diffuse plasmid throughout the eye, with no nuclear rhodamine localization. Subretinally treated control animals showed plasmid in the SRS. Conclusions: These results show that rhodamine–conjugated plasmids are effective for in vitro and in vivo gene delivery tracking and allow for gene expression. Following intravitreal injection, electroporated plasmid migrated across the plasma membrane of cells in the retinal ganglion cell layer and localized to their nuclei. After subretinal injection, plasmid was detected in the SRS, RPE, inner and outer segments, and outer nuclear layer. We are currently testing several electroporation parameters in vivo using rhodamine–conjugated plasmids.
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