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C. Chou, K. M. Janisch, J. M. Kasanuki, C. S. Lin, R. J. Davis, J. Tosi, N. K. Wang, S. H. Tsang; Red Fluorescent Protein as a Marker for Stem Cell Therapy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4536.
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Retinal pigment epithelium (RPE) is a monolayer tissue that provides structural support and nourishment to the retina. Several degenerative diseases such as age-related macular degeneration, Lebers Congenital Amaurosis and retinitis pigmentosa have significant public health consequences, and their pathology involve the dysfunction or loss of RPE cells. Therefore, stem cell therapy to treat diseases with RPE-involvement is of important clinical value. A retinal pigment epithelium specific fluorescent marker may be a useful tool in the tracking of transplanted stem cell and high-throughput screening of putative small molecules that promote stem cell to RPE differentiation. Rpe65 is expressed only in the RPE and a fluorescent protein placed under the regulation of the Rpe65 promoter may serve as a useful model for the study of stem cell to RPE differentiation.
Using the bacterial artificial chromosome recombineering method, mCherry, a second generation monomeric red fluorescent protein has been cloned under the regulation of the Rpe65 promoter. The neomycin resistance cassette was also incorporated. The transgenic construct has been electroporated into embryonic stem (ES) cells of C57/BL6. Electroporated ES cells were then grown on PA6 feeder cells in α-MEM supplemented with dexamethasone, cholera toxin and basic fibroblast growth factor to promote RPE differentiation. Fluorescence microscopy was subsequently employed used to screen for cells expressing the mCherry protein.
Fluorescence has only been observed in cells eletroporated with the recombinant construct and no fluorescence was seen in non-transduced cells.
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