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I.V. Malyukova, E.F. Wawrousek, S. Swaminathan, S.K. Sharan, S.I. Tomarev; Transgenic Mice Containing BAC Clone With a Point Mutation in the Myocilin Gene as a Model for Glaucoma . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1112.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To develop a novel mouse model of glaucoma. Methods: Full length mouse myocilin cDNA was cloned into the p3XFLAG-CMV-14 vector. Point mutations in the mouse myocilin cDNA were produced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). A point mutation in a BAC, containing the full length myocilin gene, was generated using oligonucleotide-based mutagenesis in E. coli. The myocilin gene copy number in transgenic mice containing mutated BAC DNA was estimated by real-time PCR. The distribution of wild-type and mutated myocilin proteins was investigated in western blotting experiments with antibodies raised against mouse myocilin. Results:The Tyr423His and Ile463Ser point mutations in mouse myocilin corresponded to the Tyr437His and Ile477Ser point mutations in human Myocilin. These two mutations in the human myocilin gene lead to the most severe glaucoma phenotype. Similarly to the human mutants, mutated forms of mouse myocilin were not secreted from transfected COS7 cells. Moreover, presence of the mutated mouse proteins prevented secretion of wild-type myocilin from transfected cells. The Tyr423His point mutation was introduced into BAC clone 16652 This BAC clone contained the full-length mouse myocilin gene as well as 20 kb of 5’-flanking and 85.5 kb of 3’-flanking sequences. Preliminary experiments demonstrated that the BAC 16652 transgene reproduced the expression pattern of the endogenous myocilin gene. Two lines of transgenic mice containing mutated BAC DNA 16652 were produced. The presence of the mutation in transgenic mice was confirmed by restriction enzyme analysis and by sequencing. One transgenic line contained about 30 copies, while the other line contained 2-3 copies of the mutated myocilin gene. The intracellular distribution of myocilin was different in the eyes of transgenic mice compared with its distribution in non-transgenic siblings. A morphological characterization of the eyes of transgenic mice as well as detailed characterization of the myocilin distribution in the eyes of transgenic mice is in progress. Conclusions: Since the Tyr437His mutation in the human myocilin gene leads to juvenile open angle glaucoma with elevated intraocular pressure, expression of the mutated form of mouse myocilin in the ocular tissues of transgenic mice may lead to an elevation of intraocular pressure and morphological changes similar to those in glaucoma in humans.
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