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C. J. Johnson, V. Raman, J. M. Nickerson; Electroporation Following Transcorneal Subretinal Injection of a Red Fluorescent Expression Vector in the Adult Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5341.
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Electroporation has been shown to deliver DNA to the mouse retina in vivo. In the preceding injection a needle is introduced to the subretinal space through an incision in the sclera. Unfortunately, the sclerotomy can cause intraocular bleeding, and the resulting tissue autofluorescence can mask fluorescent protein expression. We sought to investigate if the transcorneal subretinal injection would resolve the surgical complications of the transscleral method and to optimize the subsequent electroporation treatment for use in future gene delivery of RPE-specific therapeutic DNA.
Using a 34-gauge beveled needle, a puncture was made in the cornea or sclera of adult mice. A 35-gauge blunt needle was inserted into the respective incision and advanced to the subretinal space. Each surgical technique was recorded by video microscopy. For expression analysis eyes were injected with a red fluorescent (RFP) expression vector and received transscleral electroporation after injection. Control eyes lacked one or more of the following treatments: injection, plasmid, or electroporation. To mark the site of injected material eyes were injected with Quantum Dots. All eyes were harvested up to 9 days post-treatment and cryosectioned. Fluorescence expression was observed by microscopy, apoptosis detected by TUNEL assay, and morphology analyzed by H&E staining.
Eyes injected transcorneally had a lower incidence of hemorrhage than eyes incised at the sclera. RFP was observed in eyes injected through the cornea, but not in transsclerally-injected eyes or in any controls. TUNEL showed most retinal cell death near hemorrhages of transclerally-injected eyes, and H&E confirmed the presence of red blood cells at these sites. As shown in video recordings, a subretinal bleb was seen in successfully injected eyes and the location of Quantum Dots in tissue sections confirmed this assessment.
We demonstrate that our RFP expression vector can be expressed in the adult mouse retina by in vivo electroporation, and have shown the utility of the transcorneal subretinal injection over the transscleral method.
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