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S. Sarfare, G. R. Rubin, J. D. Rockwell, T. W. Kraft, S. J. Pittler; Characterization of Transgenic Mice Overexpressing GARP2 in the Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5428.
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Previous work by our laboratory showed that a knockout mouse deleted for the rod cGMP-gated cation channel β-subunit and related GARP proteins (KO) exhibits ectopic disk morphogenesis, loss of structural integrity and progressive retinal degeneration. To examine the role of GARP2 we generated transgenic mice overexpressing this protein in rod photoreceptors.
A murine GARP2 transgene construct was generated by PCR amplification of the entire GARP2 gene fused in-frame to a c-myc tag under the control of a rhodopsin promoter and flanked by the polyadenylation sequence of the mouse protamine gene. Five transgenic lines were confirmed by PCR-based genotyping, and the line expressing GARP2-myc at the highest level was chosen for further analysis. Light and EM microscopy, Westerns and functional analysis by ERG were performed using established protocols.
Quantitative analysis of GARP2 levels in the five lines demonstrated that one line expressed the protein at levels 7-10 fold greater than wild type. On the KO background the transgene led to a more severe phenotype than the KO alone, with greater outer segment disorganization and more rapid cell loss. On the WT background structural changes involving aberrant membrane growth were apparent, but degeneration was very slow. As early as one month ROS are 75% of wild-type length, however there are no gross abnormalities in the arrangement of the retinal cell layers. Dark-adapted ERG a-wave was 58% and b-wave was 69% that of wild-type at 1 month. Robust, albeit up to 50% attenuated rod ERGs were apparent in animals as old as 9 months.
Overexpression of GARP2 on the KO background does not compensate for Cngb1 KO but rather accelerates the degeneration. The similarity of the structural phenotypes generated by overexpression of GARP2 on WT and KO backgrounds indicates a role for GARP2 in maintaining the structural integrity of the ROS.
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