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Nicole R. Congrove, Christina L. Decatur, J. Brett Stanton, Alan D. Marmorstein, Brian S. McKay; Characterization of Transgenic Mouse RPE Cultures. Invest. Ophthalmol. Vis. Sci. 2011;52(14):931.
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© ARVO (1962-2015); The Authors (2016-present)
Mouse models of retinal diseases are commonly used to investigate RPE function and pathophysiology. For some types of assays, cultured RPE cells from transgenic mice would be of significant utility. Here we examine mouse RPE cultures to determine conditions under which they expressed ‘tissue-type’ differentiated properties.
We developed mouse RPE cultures from several transgenic lines by adapting methods developed by Hu and Bok (Mol Vis. 2001). In particular, early experiments illustrated that explants of the cells in CEM, as described for fetal human RPE, was particularly suited for development of ‘tissue-type’ monolayers. Cultures were analyzed by western blot analysis, immunofluorescence microscopy, and ELISA for expression of tyrosinase, pigmentation, CRABP-1, Bestrophin-1, and PEDF under the control of OA1.
Cultured mouse RPE produced monolayers of hexagonal cells that were stable for greater than 1 year. RPE capable of pigmentation (not from albino mice) were heavily pigmented within 1 month, and maintained pigment until harvest. Monolayers from all lines expressed CRABP-1, PEDF, and Bestropin-1, with the exception of the RPE from Bestrophin-1 knock-out mice. Endogenous PEDF expression levels of the mouse monolayers were similar to monolayers of fetal human RPE, and were tied to the OA1 autocrine loop (Lopez, et al. 2008). We found the best age for production of RPE monolayers to be approximately 30 day old mice.
The CEM developed for growth and differentiation of fetal human RPE works well with RPE explants from young adult mice. As we have previously described for human RPE cultures (Rak 2006), RPE marker expression in mouse RPE monolayers develops slowly over time. For example, Bestrophin-1 expression was observed in mouse RPE cultures maintained at confluence approximately 1 year, in agreement with our previous results from human RPE indicating no Bestrophin-1 expression at the 4 month time point. Mouse RPE cultures, produced and maintained using these methods, make an excellent model to study RPE function from transgenic animals.
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