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CE Schaub, S Caballero, EA Ellis, LC Shaw, MB Grant; Intraocular Injection of Plasmid DNA into an Oxygen Induced Mouse Model of Retinopathy of Prematurity . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3713.
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Purpose:Retinal neovascularization is associated with the disease state retinopathy of prematurity (ROP). We are using an oxygen induced ROP mouse model (Smith, et al. 1994) to study pathological retinal angiogenesis resulting from hypoxia. We intraocularly inject plasmid constructs encoding genes expressing either hammerhead ribozymes or proteins to determine their effect on angiogenesis in this model. The major problem to overcome is achieving adequate gene expression during the 17-day time course of the model. Our goal was to determine the optimum form of the plasmid construct to inject and the optimum time of injection to yield maximum expression of our genes in the ROP mouse model. Methods:To determine the optimum formulation of plasmid for injection, a plasmid expressing the green fluorescent protein (GFP), was either packaged into a lipsome/transferrin mixture or suspended in HEPES buffer and injected intraocularly into 1-day old mice. Mice were sacrificed on days 2-19, and the retina were removed and flat-mounted. To determine the optimum time for plasmid injection 1, 4, 7 and 8-day old mice were injected with the GFP expressing plasmid suspended in HEPES buffer. These mice were placed into high oxygen from day 7 to day 12. Mice were sacrifice on day 17 and the retina were removed and flat-mounted for fluorescence microscopy and determination of GFP expression. Results:Our analysis showed that injection of either the plasmid packaged in the liposome/transferrin mixture or the naked DNA resulted in significant transfection of retinal cells. However, the more efficient transfection of retinal cells occurred with the plasmid packaged in the liposome/transferrin mixture transfected. We also found that after the injection of plasmid on day 1, GFP expression was initiated on day 11 and continues through day 19. In addition, we found that after the initiation of angiogenesis on day 12, the expression of GFP in the ROP mouse model increases from day 12 to day 17. Conclusion:Our results demonstrate that the intraocular injection of plasmid, regardless of formulation, results in the expression GFP within the time course of the ROP mouse model, but the plasmid packaged in the liposome/transferrin mixture is more efficient at transfecting cells in vivo than is plasmid suspended in HEPES buffer.
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