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Delores A Davis, Marci L Smith, Youwen Zhang, Andrew F X Goldberg, Steven J Pittler; Absence of GARP2 in mice leads to slowly progressive structural deficits in rod photoreceptors. Invest. Ophthalmol. Vis. Sci. 2014;55(13):406.
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© ARVO (1962-2015); The Authors (2016-present)
The Cngb1 locus encodes the beta subunit of the rod photoreceptor CNG channel, and by alternative splicing two soluble glutamic acid rich proteins, GARP1 and GARP2. We previously reported that loss of all Cngb1-encoded proteins alters disk morphogenesis and structural integrity and attenuates the rod photoresponse, and ultimately leads to photoreceptor death and blindness (Cngb1-X1; Zhang et al. 2009 J. Cell Sci. 122:1192). Here we used genome-editing technology to selectively delete expression of GARP2 to singularly assess its role in the rods.
Using Sigma’s CompoZr® ZFN technology, exon 12a that is unique to GARP2 was targeted by injection of ZFN RNA into the male pronucleus of single cell fertilized mouse embryos that were allowed to develop using standard mouse transgene techniques. One allele was identified that contained a complete deletion of exon 12a and upstream acceptor site that represents a true GARP2 null. Established procedures for OCT, light microscopy, EM and Western analysis were used to analyze the GARP2 KO retina compared to WT.
Western analysis of total retina protein demonstrated the absence of 73 kD murine GARP2 in the knockout retina that is abundant in WT mouse retina, however beta-subunit and GARP1 expression were unaffected. OCT imaging of 1-6 month old mouse retina showed no distinct differences in retinal layer thickness but changes in hyperreflectivity were apparent in the outer segment region. By light microscopy, as early as 2 months rod outer segments (ROS) are less uniform and the ROS frequently extend parallel to the RPE. At this time point in EM micrographs ROS disks appear normal in all rods examined. By 8.5 months a 20% loss of nuclei in the ONL was apparent and ROS appear shorter.
GARP2 knockout alone exhibits a more subtle phenotype than was observed in the Cngb1-X1 total photoreceptor null mouse. GARP2 does not appear to be required for disk morphogenesis, however it is necessary to maintain ROS structural integrity as the animal matures and in aging mice its absence leads to retinal degeneration. Thus, the GARP2 KO model may represent a good model for the study of later onset retinal degeneration.
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