PMID: 9418739Jan 7, 1998Paper

Expression and upregulation of microtubule-associated protein 1B in cultured retinal pigment epithelial cells

Investigative Ophthalmology & Visual Science
P EsserM Weller

Abstract

During routine cell culture and under pathologic conditions, human retinal pigment epithelial (RPE) cells lose epithelial characteristics and change their morphology. In this study, changes in gene expression in RPE cells of different generations were evaluated by polymerase-chain-reaction-based differential display mRNA analysis (DD-RT-PCR). Total RNA was prepared from freshly isolated and cultured human RPE cells of passages P0 and P3 and was subjected to DD-RT-PCR. One band with enhanced expression was excised, reamplified, and partially sequenced, using a modified dideoxy chain termination approach. Expression of the corresponding protein was ascertained by immunocytochemical analysis. Differential display RT-PCR showed enhanced expression of a specific RNA in P3 cells compared with that in P0 cells. Sequence alignment revealed 98% identity with the 3' end of the coding sequence of human microtubule-associated protein 1B (MAP1B). Confirmation of induced expression of MAP1B mRNA was obtained by PCR with specific primers and by immunocytochemical analysis in cultured RPE cells and in surgically removed epiretinal membranes from patients with proliferative vitreoretinopathy. No expression of MAP1B mRNA or protein was detected ...Continue Reading

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