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R. Gupta, C. Asomugha, O. P. Srivastava; Characterization of a Transgenic Mouse Model Expressing Human Deamidated A-Crystallin. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4616.
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The purpose of the study was to characterize biochemical and morphological changes in transgenic mice lenses that were expressing deamidated αA-crystallin.
Transgenic mice expressing deamidated Asn at 101 residue in αA-crystallin were generated using mouse αA-crystallin promoter. Wild type (WT) αA-crystallin encoding amino terminal His-tagged cDNA was used to differentiate transgene expression from endogenous mouse αA-crystallin. Deamidation of Asn to Asp was introduced using a QuikChange site-directed mutagenesis kit (Stratagene). A transgenic mouse expressing WTαA-crystallin was used as a control (a gift from Dr. Mark Petrash, University of Colorado, Denver). Biochemical properties of crystallins from both deamidated and WT mouse lenses were studied using gel filtration, 2D-DIGE gel electrophoresis, and immunohistochemical staining using a monoclonal antibody to the His-tag region (Novagen). Gross lens and fiber cell morphology were visualized with a digital camera attached to a stereo microscope and with Zeiss Axio Plan 2 imaging system, respectively.
RT-PCR confirmed the transcription of transgene (N101D mutant) and western blot/immunohistochemical staining confirmed the expression of the transgene in all independent founder lines compared to non-transgenic animals. Deamidated littermates showed relatively slow growth and slight decrease in lens weight compared to WT littermates. The water soluble (WS)- protein content decreased with concomitant increase in water insoluble (WI)- proteins in deamidated lenses compared to WT lenses. Following size-exclusion chromatography of WS- proteins, the deamidated lenses showed relatively decreased amounts of α- and β- crystallins compared to WT lenses. Relative to WT, the deamidated animals showed altered packing of fiber cells with aging. Further, a membrane protein, AQP0 was detected in higher levels in WI fraction of deamidated lens compared to WT lenses.
Expression of mutant N101D αA-crystallin results in higher level of WI-protein and altered packing of fiber cells in the transgenic mice lenses compared to WT mice.
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