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J Ueda, Y Li, MS Goh, Y Maruyama, J Sugar, BY J T Yue; A Reporter Gene Construct Containing 1.4-kb Alpha1-proteinase Inhibitor Promoter Confers Expression In The Cornea of Transgenic Mice . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3227.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The alpha1-proteinase inhibitor (a1-PI) gene has been shown to be markedly downregulated in keratoconus corneas. We cloned and sequenced a 1.4-kb promoter fragment of the human a1-PI gene. This fragment was functional specifically in human corneal cells in transient transfection experiments. To extend the in vitro experimentation to in vivo, transgenic mice were produced in which the spatial and temporal expression of the a1-PI promoter was investigated. Methods: A 1.4-kb human a1-PI 5'-flanking sequence fused to the E. coli LacZ gene was used to generate transgenic mice. The corneas and various other tissues of transgenic and non-transgenic mice were immunostained with a rabbit polyclonal anti-E. coli b-galactosidase antibody. RT-PCR analyses of LacZ were also performed using RNAs extracted from the various tissues. For developmental studies, tissues from embryonic (E) and postnatal (P) transgenic mice on days E10.5, E12.5, E15.5, E18.5, P0, P7, P11 and P30 were examined immunohistochemically. Results: In transgenic mice, positive b-galactosidase staining was observed in all layers of the corneal epithelium and stroma. No staining was found in other ocular tissues, liver or kidney. Moderate to weak expression was noted in the skin, brain and white blood cells. Developmental studies indicated that the a1-PI promoter-driven LacZ expression in the corneal epithelium began at E15.5 and remained constant after E18.5. The expression in the stroma started on P7 and was the most prominent on P11. Conclusion: The 1.4-kb a1-PI fragment was found to target LacZ expression preferentially to the epithelium and stroma of the mouse cornea. This promoter may offer an option for targeting foreign genes to the cornea in future transgenic experiments for studies of epithelial-stromal interactions and diseases such as keratoconus.
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