| PURPOSE. To generate a transgenic zebrafish model of retinitis pigmentosa (RP), in which the retinal degeneration was induced by rhodopsin double mutation-R135G/G188R, and to investigate the characteristics of its retinal degeneration.METHODS. Transient transgenic zebrafish (FO generations) were generated via embryo microinjection with genetically engineered pXOP1.3-EGFP-rho and pXOP1.3-EGFP-rho R135G/G188R plasmids that expressed the wild-type zebrafish rhodopsin or its double mutants, respectively. Those EGFP-positive FO generation transgenic larvae were screened out via in vivo conventional fluorescence microscope observation. Using fluorescence or immunofluorescence cryosection assay, transgenic rhodopsin levels and its localizations in the retina, as well as the characteristics of retinal degeneration was determined by fluorescence microscopy.RESULTS. The1.3-kb Xenopus rhodopsin promoter fragment instead of the whole5.5-kb XOP was sufficient to promote the expression of our transgenic constructs. And it was possible to identify the transgenic animals by obtaining fluorescence images of the eyes of intact, living zebrafish larvae under conventional fluorescence microscope:during three rounds of injections, an approximate5%-7%mean EGFP-positive rate was obtained in about1000embryos. On cryosection assay, Rhodopsin-R135G/G188R expression levels decreased by the time of5dpf as opposed to that of the control transgenic rhodopsin, which obviously increased. Along with the retinal EGFP distribution feature, this observation suggested that the double mutated rhodopsin might have induced rod-specific retinal degeneration; whereas the control transgenic rhodopsin did not, indicating that rod photoreceptor death was specific to the R135G/G188R mutation.CONCLUSIONS. Using plasmids consisting of cell-specific promoter plus EGFP CDS tagged double mutant of the Rho CDS, a transient transgenic zebrafish RP model was generated. This model verified that the1.3kb XOP fragment was a sufficient rod-specific promoter across species, and supported a role for the possibility that Class â…¡ rhodopsin mutation-R135G/G188R caused misfolding in RP with a phenotype of rod death. Meanwhile, these zebrafish implicated the potential of being used as models of human inherited retinitis pigmentosa and the utility of EGFP-Rho fusion protein as a probe for discovering the underlying pathogenic mechanisms of this disease, or even being used as experimental materials to explore its gene therapy. |
