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Beatrice Tam, Jonathan Lin, Orson Moritz; An Xbp1-GFP-rhodopsin reporter transgene for assessing ER stress in Xenopus laevis models of retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2013;54(15):282.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated that in transgenic X. laevis, P23H rhodopsin is significantly retained in the ER, and that light exposure exacerbates this ER retention and promotes retinal degeneration. Here we examine whether accumulation of the mutant protein induces ER stress in vivo by monitoring the splicing of an Xbp1-GFP reporter construct.
The DNA binding domain of murine Xbp1 was deleted and an out of frame cDNA encoding a GFP-rhodopsin fusion protein inserted downstream of the unconventional splice site. Upon ER stress, splicing of the mRNA produces an in-frame Xbp1-GFP-rhodopsin (XGR) fusion protein. This engineered cDNA was used to generate transgenic X. laevis expressing the fusion protein under the control of the opsin promoter. An XGR frog was mated with a transgenic frog expressing bovine P23H rhodopsin (bRhoP23H). The resulting tadpoles were raised for 14 days in constant dark, which prevents retinal degeneration in this animal model. Half the tadpoles were then exposed to two 12:12 light:dark cycles and returned to the dark. The other half were maintained in constant dark. On day 21, all tadpoles were killed and their eyes used for dot blot analysis or for fluorescence microscopy.
In our line of XGR animals, GFP fluorescence peaked shortly after the onset of rod opsin expression (~d5) and thereafter declined and was virtually undetectable by d21. Interestingly, XGR distributed to both inner and outer segment membranes. In contrast, a similar protein (GFP-RhoCT44) which does not include Xbp1, is almost exclusively delivered to the outer segment. In cryosections of eyes that were not expressing bRhoP23H, little or no GFP fluorescence was observed regardless of whether the animals had received light exposure or not. Similarly, dark reared bRhoP23H retinas expressed minimal XGR. In contrast, light exposed bRhoP23H retinas exhibited higher levels of fluorescence. Furthermore, nuclear XGR localization was observed in this group. As expected, retinas expressing bRhoP23H underwent retinal degeneration when exposed to light.
At d21, significant levels of Xbp-GFP-RhoCT44 were observed only in light exposed animals expressing bRhoP23H. This is consistent with a pathogenic role for light-induced P23H rhodopsin misfolding and ER stress in this model of retinitis pigmentosa.
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