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I.V. Malyukova, V.V. Senatorov, E.F. Wawrousek, S. Swaminathan, S.K. Sharan, H. Yang, M.A. Avila, M.M. Civan, S.I. Tomarev; Presence of mutant myocilin gene in mice doesn’t lead to IOP elevation. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4372.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To evaluate relationship between intraocular pressure and presence of mutant myocilin gene in mice Methods: The Tyr423His point mutation was introduced into a BAC clone containing the full length mouse myocilin gene, using oligonucleotide–based recombining in E. coli. This BAC DNA was used to generate transgenic mice. The myocilin gene copy number in transgenic mice was estimated by real–time PCR. The distribution of myocilin in the eyes of transgenic and wild–type littermates was investigated in western blotting experiments and by immunohistochemistry with antibodies raised against mouse myocilin. Intraocular pressure (IOP) was measured with the servo–null micropipette system after animals were anesthetized with a mixture of ketamine and xylazine. Results: 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. The Tyr423His point mutation in mouse myocilin corresponded to the Tyr437His point mutations in human Myocilin which may lead to severe juvenile open–angle glaucoma. Four lines of transgenic mice were generated. Line #18, containing 2–3 copies of the mutated myocilin gene, was selected for further analysis. Myocilin was present mainly in the trabecular meshwork and sclera of 6–8 month old transgenic and wild–type siblings as judged by immunostaining of eye sections with antibodies against mouse myocilin. Immunofluorescent staining was 2–3 fold higher in transgenic mice compared with wild–type littermates in both tissues. Western blot analysis demonstrated that myocilin was present mainly in the soluble fraction in wild–type animals and in the pellet fraction in transgenic mice. In situ hybridization and real–time PCR analysis showed no pronounced changes in myocilin mRNA levels in transgenic mice compared with wild–type littermates. IOP was 10.1±0.7 mm Hg (14 eyes/7 mice) and 11.1±0.7 mm Hg (16 eyes/8 mice) for 11–12 month old transgenic and wild–type littermates, respectively, under ketamine–xylazine anesthesia. Experiments are in progress to evaluate relationships between intraocular pressure and expression of myocilin in three other lines of transgenic mice. Conclusions: The presence of mutated Tyr423His myocilin gene in the eyes of transgenic heterozygous line #18 does not lead to statistically significant changes in IOP at age 11–12 months.
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