Expression of human ABCR in Xenopus laevis photoreceptors

Abstract

Abstract: : Purpose: ABCR (ABCA4) is a photoreceptor–specific member of the ATP–binding cassette (ABC) family, and mutations of ABCR are associated with many retinal phenotypes including Stargardt disease, Fundus Flavimaculatus, combined cone–rod dystrophy and retinitis pigmentosa. Heterozygous mutations have also been associated with the multi–factorial disorder age–related macular degeneration (ARMD). Our purpose is to develop an experimental system for investigating mechanisms regulating intracellular trafficking of ABCR and for testing mutations found in domains involved in cellular localization of ABCR. Methods: Our approach is to express a human ABCR transgene in photoreceptors of X. laevis tadpoles. DNA fragments containing both wild type and mutated (C75G, S100P, C2150R and D2177N) ABCR coding sequence were cloned into the pXOP vector containing the promoter for the X. laevis rhodopsin gene, and used for transgenesis. Constructs with C–terminal epitope tags, including the 1–D4 epitope from mammalian rhodopsin, were also examined. The retinas of 2–4 week old tadpoles were studied for the expression and localization of transgenic ABCR by immunofluorescence methods. Results: Expression was restricted to photoreceptor cells, and was variable from cell to cell, as observed previously for transgenes with the pXOP promoter. In some cells very bright staining of the rims of the disks was observed in rod outer segments. Conclusions: Human ABCR can be expressed from a transgene in Xenopus photoreceptors. At least some of it appears to be properly transported to outer segments and to be properly localized to disk rims. The transgenic Xenopus approach is very promising for unraveling the mechanisms for ABCR trafficking and localization, and for assaying functional consequences of mutations found in human patients.

Keywords: photoreceptors • gene/expression • transgenics/knock–outs