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J. Zhang, H. Liu, Y. Zhang, M.-T. Nguyen, M. Call, C.-Y. Liu, W. W. Kao; Characterization of a Tet-On Inducible, Pax6 Promoter Driven Transgenic Mouse Line. Invest. Ophthalmol. Vis. Sci. 2010;51(13):350.
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© ARVO (1962-2015); The Authors (2016-present)
To characterize a new tet-on transgenic mouse line utilizing the Pax6 promoter-rtTA transgene (P6R) and the dual-reporter ROSA26mTmG line.
To characterize Pax6-rtTA (P6R), we crossed this P6R line with ROSA26mTmG and tet-O-Cre (TC) transgenic mice to prepare triple P6R/TC/Rosa26mTmG transgenic mice, which will allow the identification of Pax6 expressing cells by the expression of a membrane bound enhanced green fluorescent protein (mG). Once the cell has undergone homologous recombination, EGFP expression will be maintained via the chicken beta-actin promoter that is targeted into the Rosa26 allele and is no longer dependent of the Pax6 gene. After initial screening of the founders, P6R/TC/ROSA26mTmG mice were induced at various times in development (E8.5, E16.5) by either a pulse dose of doxycycline or by continuous induction in the chow. In addition, transgenic mice were also induced from P10 to P21 to assess the postnatal expression pattern of Pax6 within ocular surface tissues. In all cases, ocular surface tissues were harvested at P21 and examined by fluorescence microscopy. All reported research was conducted in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research.
Fluorescent microscopy has revealed two lines of P6R with varying expression patterns. In the first line, mG expression is restricted to a subset of corneal endothelial cells but is absent from the corneal epithelium. This expression pattern was observed with induction at E16.5 as well as P10. The second line expressed mG in a pattern that coincides with endogenous expression of Pax6; that is within the epithelial layers of several ocular surface tissues (cornea, conjunctiva).
Characterization of the P6R line has revealed a very promising tool for the study of ocular surface morphogenesis. While more work needs to be done to further characterize this new transgenic line; therein lies a unique tool to genetically manipulate various populations of cells in a spatial and temporal manner.
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