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W. Wang, P. Gonzalez, P.B. Liton, M. Caballero, D.L. Epstein; Effects of mutant TIGR/myocilin expression in trabecular meshwork cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3660.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To generate recombinant adenoviral vectors expressing TIGR/myocilin mutations to investigate possible mechanisms through which myocilin (Myoc) mutations might contribute to the pathogenesis of glaucoma. Methods: PCR–directed mutagenesis was used to generate three cDNAs, each containing one of three known Myoc mutations associated with primary open angle glaucoma (POAG). These mutations, Pro370Leu, Thr377Met, and Gln368Stop have reported mean ages of disease onset of 12, 37, and 59 years, respectively. The cDNAs carrying the Myoc mutations were used to construct three replication deficient recombinant adenoviruses, each under the control of the CMV promoter using Stratagene’s AdEasy XL adenoviral vector system. The three viruses were then used to infect trabecular meshwork (TM) cell cultures, and viral gene expression was tested via Western blotting (WB) using an anti–Myoc specific antibody. Results: TM cells infected with the Pro370Leu and Thr377Met mutations each showed a single prominent band at 55kDa, corresponding to the previously reported size of the wild type Myoc protein. In addition, the Thr377Met mutation–infected cells also showed several faster migrating bands that could represent Myoc protein degradation products. The cells infected with the Gln368Stop mutation showed a single band that corresponded to the truncated form of the Myoc protein. Conclusions: The availability of these three recombinant adenoviral vectors carrying known Myoc mutations represents a new tool to study the mechanisms through which expression of Myoc mutations contribute to glaucoma pathogenesis both in–vitro and in–vivo.
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