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KJ Wahlin, EA Grice, AS Hackam, PA Campochiaro, DJ Zack, R Adler; Analysis of Gene Expression in Isolated, Microcaptured Mouse Photoreceptor and Müller Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):669.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In retinal degeneration animal models, photoreceptor rescue by intraocularly injected neurotrophic factors appears to occur through indirect mechanisms, likely mediated by Müller cells (Wahlin et al, 2000, 2001). It is therefore important to identify putative trophic factor-induced Müller cell products that could provide new therapeutic tools for treatment of these blinding diseases. Towards this long-term goal, the purpose of the present study was to develop methods for generating cDNAs from isolated Müller cells and photoreceptors. Methods: Individual cells from papain-dissociated mouse retinae were captured with micropipettes under slight negative pressure, and identified morphologically and by rhodopsin and CRALBP immunocytochemistry. After cell lysis and reverse transcription, first strand cDNA from each cell was polyA-tailed, and PCR-amplified using oligo-d(T). Gel electrophoresis and PCR were used to compare cDNAs from freshly isolated, paraformaldehyde-fixed, and fixed and immunostained cells. Results: Dissociated rod and Müller glia cells maintained their characteristic morphology, which correlated well with their immunocytochemical properties. Abundant cDNA could be synthesized from each individual cell, even after fixation (which allows harvesting large number of cells from each retina) and immunostaining (which corroborates their identity). Cell-specific gene products were detectable by PCR in all cases, although with some differences between differently processed cells. Conclusion: Gene expression can be analyzed in cDNAs from isolated, identified mouse retinal photoreceptors and Müller cells. Subtractive and/or macroarray approaches can be applied to these cDNAs to compare patterns of gene expression between trophic factor-treated and untreated Müller and photoreceptor cells.
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