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F.L. Kernan, P.F. Kenna, M. Miura, P. Humphries, G.J. Farrar; Development of a Strategy for the Evaluation of the Therapeutic Potential of p35 for Retinal Degenerations . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1683.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop a strategy, which will enable the evaluation of the therapeutic potential of p35 for retinal disorders such as retinitis pigmentosa (RP), using a loxP–p35 transgenic mouse model. Methods: p35 is a baculoviral caspase inhibitor that has been shown to inhibit induced apoptosis in a line of cone photoreceptor 661W cells. The anti–apoptotic effects of p35 have now been optimised in these cells. Cre recombinase–mediated activation of the p35 gene was evaluated using retinal explants. Two constructs with either rod or cone photoreceptor specific promoters driving expression of Cre (iCre) were generated. Tissue specificity and functionality of these constructs were evaluated by transient transfection in cell culture followed by electroporation into retinal explants. Results: The functionality of iCre constructs was demonstrated using a ß gal/neo reporter in 293T cells and by electroporation of retinal explants. The iCre constructs were electroporated into retinal explants of transgenic p35 mice and p35 expression was evaluated by immunocytochemistry. Conclusions: Retinal explants have been used initially to validate photoreceptor specific expression of Cre recombinase constructs. These constructs were then deomonstrated to produce tissue specific expression of p35 in photoreceptor cells of retinal explants from p35 transgenic mice. In principle this strategy will now be used to explore the therapeutic potential of p35 in vivo for retinal degenerations such as RP.
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