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J.R. Taube, M.K. Duncan; Control of High-level Crystallin Gene Expression in Lens Fiber Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1233.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: While much is known about the mechanisms controlling lens-specific gene expression, the elements controlling the high levels of crystallin expression in the lens remain elusive. The region of the promoter which drives high level expression in fiber cells has been identified in one gene, ßB1-crystallin. Transcription of the ßB1-crystallin gene results in ~2% of the mRNA in the lens. The aim of this work is to locate the elements controlling the high-level expression of the chicken ßB1–crystallin gene, and to determine if chicken lens annular pad cells are suitable for the study of fiber-cell specific gene expression. Methods: Chicken lens annular pad (CLAP) cells were cultured from 19-day embryonic chicken lenses, and transiently transfected with chicken ßB1–crystallin promoter pBasic CAT constructs. The transfection constructs were pBasic CAT, -152/+30 CAT, -245/+30 CAT, and -432/+30 CAT. SDS-PAGE and western blot analysis was done on protein extracts from annular pads, fibers, and lens epithelium from 19-day embryonic chicken lenses, and CLAP cells cultured for 10 days. Results: The annular pad cells and cultured CLAP cells do endogenously express ßB1–crystallin. The levels of the ßB1-crystallin protein from CLAP cells cultured for 10 days are similar to those of the lens fiber cells. The -245/+30/CAT construct was the most highly expressed in the transfections of CLAP cells. This is in contrast to our results in transgenic mouse lenses, where the -432/+30/CAT construct expresses the highest, at levels equivalent to the endogenous gene (1-2 % of mRNA). Also these results differ from those obtained in transfections of lens epithelial cells (which do not endogenously express ßB1–crystallin), which express the -152/+30/CAT construct the most. Conclusions: Transiently transfected CLAP cells, cultured from 19-day embryonic chicken lenses, are suitable to study fiber-cell specific elements. Differences in the results obtained in transiently transfected CLAP cells and the lens fiber cells of transgenic mice are likely to be due to the effects of chromatin structure. The high-level expression of the chicken ßB1–crystallin promoter is clearly controlled by multiple transcription elements within the region -432/-152.
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