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Hanane A. Koteiche, Wenbiao Chen, David M. Anderson, Kevin L. Schey, Hassane S. Mchaourab; Zebrafish Models of α-Crystallin Mutants Linked to Human Cataract. Invest. Ophthalmol. Vis. Sci. 2011;52(14):801.
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A number of αA-crystallin mutants have been linked to autosomal cataracts in humans. While studies in vitro have demonstrated disregulation of the chaperone activity in these mutants, the consequences on molecular interactions in vivo have yet to be defined. To elucidate the impact of these mutants on lens transparency, we have created transgenic zebrafish (Danio Rerio) that expresses the two αA-crystallin mutants R49C and R116C.
We constructed Tol2 transposon-based transgenes that express the cDNA of the wild-type or mutant αA-crystallin under the control of zebrafish αA-crystallin promoter (-0.7αcrystallin:αAcrystallin). We also included in the transgene a (cmlc2:mCerulean) that fluorescently marks cardiac myocytes for identification of transgenic fish. For MALDI imaging, zebrafish head regions were cryosectioned and tissue sections were coated with MALDI matrix either by sublimation of 2,5-dihydroxybenzoic acid (for lipids) or by spraying sinapinic acid (proteins). Images were acquired with 10-40 micron resolution using a Bruker AutFlex MALDI-TOF instrument. Images were assembled in Bruker FlexImaging software. Lipids were identified by accurate mass measurement and tandem mass spectrometry.
The transgenes were injected together with RNA encoding the Tol2 transposase. Injected fish with fluorescent cardiomyocytes were analysed or raised to maturity. The progeny of the injected fish with fluorescent heart will be maitained as stable transgenic lines. Embryos (3-7 dpf) expressing human WT and mutant αA-crystallins WT were examined by bright field microcsopy. Preliminary analysis reveal light scattering by the lens of embryos expressing the mutant proteins. In addition, we have begun to use MALDI mass spectrometry imaging to characterize the proteome and lipid profiles of zebrafish lenses.
Although preliminary, our results suggest that Zebrafish can be used as a model for hereditary cataracts. The transparency and relatively large eyes make zebrafish embryos an ideal model to visualize alterations in molecular interactions induced by these mutants.
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