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S. Brockbank, M. Oliver, G.M. Seigel, W.J. Curry; Proteomic analysis and characterisation of the R28 immortalized retinal precursor cell line. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5395.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Currently there is no effective treatment for diseases of the degenerating retina and prospect of viable retinal transplantation has proved difficult and problematic. The focus has now shifted to (i) studies of the inherent potential of stem/progenitor cells and (ii) subretinal microinjections of various neuronal and glial cell types. Before cell based therapy can be utilised a clearer understanding of factors that facilitate cellular differentiation, survival, and cell–cell communication need to be identified. Consequently this study aims to characterise the R28 retinal precursor cell line that expresses both neuronal and glial cell markers. Initially the study will focus on the proteome of the R28 cell line and aims to identify proteins and peptides which may be used to aid retinal cellular transplantation and stem cell technologies. Methods: R28 cells were grown to confluence on T75 culture flasks. Protein from the R28 cell line was extracted overnight at 4 °C in 8 M Urea, 20 mM DTT, 2% CHAPS, and 0.5% carrier ampholytes. The sample was centrifuged, the supernatant decanted, lyophilised and reconstituted in 1 ml 8 M Urea, 20 mM DTT, 2% CHAPS, and 0.5% carrier ampholytes and the protein estimated using PlusOne 2–D Quant Kit (Amersham). Two dimensional electrophoresis was performed using Zoom® IPG strips and Zoom® NuPAGE® Gels (Invitrogen). Results:Resultant protein spots were stained with Brilliant Blue G colloidal stain (Sigma) and with specific post–translational proteome markers (Pro–Q® Diamond Phosphoprotein and Emerald 300 Glycoprotein stains, Molecular Probes). Resolved spots were excised and subjected to tryptic digestion and processed for Q–TOF Ultima API tandem mass spectrometry (Micromass UK). Gels were analysed using Phoretix 2D software (Nonlinear Dynamics). Conclusions: Initial proteome analysis of the R28 retinal precursor cell line defined structural and metabolic proteins central to cellular integrity and survival. Further defined proteome analysis has demonstrated that this cell line will be a useful tool in the search for proteins and peptides which could be used in the fight against diseases of the degenerating retina.
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