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AM Achyuthan, EH Budi, R Elias, M Cardenas, W Cao, JF McGinnis; Regulation of the Rat Recoverin Gene in vivo and in Primary Cell Cultures . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1446.
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Purpose:To characterize the regulation of the recoverin gene in vivo and in cell culture. Methods:The rat recoverin protein was analyzed during the first three weeks of development in vivo and in culture using Western immunoblot techniques. Immunofluorescence microscopy was used to quantitate the appearance of recoverin positive and rhodopsin positive cells in culture. Rat recoverin cDNA was cloned from isolated retina RNA using RT- PCR and Invitrogen's TOPO cloning kit. A 200 bp region of the 5' untranslated end of the rat recoverin gene was amplified from genomic DNA, isolated and sequenced. The cDNA and 5' end of rat recoverin were compared to those of the human and mouse gene using the Multalin program. Results: The developmental appearance of recoverin in culture parallels that which occurs in vivo. Recoverin positive cells appear earlier than rhodopsin positive cells and are present in higher numbers. The isolated rat recoverin DNA has 98 % identity to the mouse retinal recoverin gene and the 23kDa protein has 202 amino acids with 98.5 % identity to the mouse recoverin protein. The putative regulatory elements, identified in the mouse and human recoverin genes, are also present in the rat gene. Conclusion:The primary cell cultures of dissociated rat retina mirror the in vivo developmental expression of the recoverin gene and the presence of identical regulatory elements in the rat, mouse and human recoverin genes support the validity of the analysis of the rodent recoverin genes as models for the human gene.
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