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O.L. Moritz, G. Li, B.M. Tam, M. Potter; Electrophysiological Findings in Transgenic X. Laevis Expressing P23H Rhodopsin . Invest. Ophthalmol. Vis. Sci. 2006;47(13):806.
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© ARVO (1962-2015); The Authors (2016-present)
We have developed a system for the expression of rhodopsin mutants in X. laevis photoreceptors at levels sufficient to cause retinal degeneration. However, expression levels are variable, and the majority of primary transgenic animals do not have a retinal degeneration phenotype. Here we show that ERG analysis, which is non–invasive and provides a direct readout of photoreceptor function, allows us to identify transgenic founders with retinal degeneration phenotypes for further study. Because X. laevis have numerous cones and are long–lived, this should be a useful system for longitudinal studies examining rod–cone interactions during retinal degeneration.
Transgenic X. laevis expressing P23H rhodopsin under control of the X. laevis opsin promoter were generated using the nuclear injection method of Kroll and Amaya, and transgenic embryos were identified using G418 selection. Twenty–eight primary transgenic animals were raised to post–metamorphic stages. We recorded scotopic ERG series from these animals and a group of control animals. The recordings were made with a glass electrode connected to an LKC UTAS–2000 Ganzfeld ERG apparatus, with additional amplification in series. ERG B–waves were fit to the Naka–Rushton equation to determine Vmax values, and A–waves were fit to the Lamb and Pugh model of phototransduction (as described by Shady and Birch) to determine Rmp3 values. We also recorded photopic and flicker ERGs from wild type animals.
In plots of Rmp3 vs. Vmax, the majority of data points from transgenic animals clustered with data from wild type animals. However, we identified a group of seven animals that did not cluster with wild type. These animals showed variable phenotypes in which either the Rmp3, Vmax, or both were significantly affected. The differences in phenotypes likely reflect variable expression levels of transgenic rhodopsin resulting in variable extent of photoreceptor death.
Electroretinography is a useful method for non–invasive identification of transgenic X. laevis with retinal degeneration phenotypes. Transgenic X. laevis expressing P23H rhodopsin exhibit a range of ERG phenotypes consistent with variable transgene expression and variable extent of degeneration. Future studies will examine the effects of the P23H mutation on cone function in these animals using optimized photopic and flicker ERG protocols, and comparison of phenotypes associated with different rhodopsin mutations.
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