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J.–H. Chang, J.A. D. Javier, F.H. Casanova, D.T. Azar; MT1–MMP Expression and Promoter Activity in Transgenic Mouse Cornea . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4484.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously used in situ hybridization methods to determine the expression of MT1–MMP (MMP–14). Our previous immunolocalization experiments showed variable specificity of commercially–available and in–house anti–MT1–MMP antibodies. Our purpose was to develop a MT1–MMP transgenic mouse and characterize MT1–MMP expression and its promoter activity in the cornea. Methods: In the first phase of the current studies, we developed a MT1–MMP promoter GFP transgenic mouse for further exploration of the role of MT1–MMP in corneal wound healing and neovascularization. To confirm the promoter activities, we used transient transfection assays in immortalized corneal fibroblasts of mouse MT1–MMP promoter luciferase constructs which were generously provided by Dr. J. Madri; Yale University. Mouse fibroblasts (104) were grown in 24 well plates of DMEM medium containing 10% heat–inactivated fetal calf serum and transfected with 2ug of MT1–MMP luciferase constructs. The transfected cells were grown for 15 h, washed once with Tris–buffered saline and lysed. Luciferase activities were determined with a luminometer. MT1–MMP promoter–GFP constructs were generated, transiently expressed in NIH–3T3 cells. Heterozygote transgenic mice were generated on a 129J background. Breeding colonies were established and GFP positive mice were generated, as confirmed by Polymerase Chain Reaction. Immunolocalization of corneal GFP expression was also determined by anti–GFP antibodies. Results: Corneal fibroblast transiently transfected with MT1–MMP promoter–luciferase constructs (220, 300, 400, 760, 1050, and 3300) displayed that MT1–MMP promoter (1050) produced higher luciferase activity. Gross examination of the MT1–MMP promoter–GFP transgenic mice showed no obvious abnormalities. There were no gross abnormalities in the cornea and anterior segments. Immunolocalization confirmed the presence of GFP in the unwounded corneal epithelium and stroma. Conclusions: This is the first report of a successful attempt at generating MT1–MMP promoter GFP transgenic mice. The absence of obvious abnormalities in the corneas and the ability to detect GFP in unwounded corneal tissue suggest this animal model may be helpful in increasing our understanding of the role of MT1–MMP in corneal wound healing and ocular neovascularization in vivo.
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