Abstract
Abstract: :
Purpose:The rhodopsin mutation P23H is the most commonly occurring cause of autosomal dominant retinitis pigmentosa in the North American population. This rhodopsin mutant has been studied in cultured cells and transgenic mice, but the mechanisms by which it causes photoreceptor cell death and retinal degeneration are not understood. In order to investigate the mechanisms of cell death resulting from this mutation, we have modeled this disorder in transgenic X. laevis. Methods: Transgenes were based on the X. laevis rhodopsin cDNA and promoter, and incorporated a mutation that introduces an antibody epitope, allowing us to distinguish the resulting protein from endogenous X. laevis rhodopsin. Transgene constructs also incorporated a neomycin–resistance gene, allowing identification of transgenic animals using G418 selection. Transgenes were constructed with or without the mutation P23H, and with or without mutations designed to interfere with rhodopsin signal transduction. Large numbers of transgenic animals were generated by nuclear transplantation, and identified by G418 selection. At 14 days post fertilization, the extent of retinal degeneration was determined by assessing total rhodopsin levels using a dot blot method. Contralateral eyes were studied by confocal microscopy. Results: Retinal degeneration occurred in transgenic animals generated using the rhodopsin P23H mutation at a significantly higher rate than in control animals, as indicated by a lower total rhodopsin content in these eyes. The average expression level of the rhoP23H rhodopsin protein was also much lower than control constructs. Degeneration was confirmed by confocal microscopy, which also demonstrated mislocalization of P23H rhodopsin to inner segment membranes. Conclusions: We have developed a novel X. laevis model of retinal degeneration induced by the rhodopsin P23H mutation. Future studies will be directed at using this model to identify pathways involved in photoreceptor cell death and retinitis pigmentosa.
Keywords: retinal degenerations: cell biology • photoreceptors • transgenics/knock–outs