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D. Kojima, Y. Fukada, J.E. Dowling; Visualization of VAL-Opsin-Expressing Cells by Zebrafish Transgenesis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5112.
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Purpose: VAL-opsin is a photoreceptive protein present both in a subset of retinal horizontal cells and diencephalic cells of adult zebrafish (Kojima et al., J. Neurosci. 20: 2845-2851, 2000). We previously established three lines of transgenic zebrafish having a GFP reporter gene which was fused to a 7.2 kb 5’-flanking sequence of the zebrafish VAL-opsin gene, but GFP expression in these transgenic lines was too weak to be visualized with fluorescence. The aim of this study is to visualize the VAL-opsin-expressing cells with GFP fluorescence by using the Gal4VP16-UAS system, which should amplify the GFP expression driven by the VAL-opsin promoter. Methods: An expression vector, iV7-GVP-UG, was constructed from two components: a transcriptional activator component (the 7.2 kb VAL-opsin promoter and Gal4VP16 gene) and an effector component (Gal4-binding UAS sites, basal promoter and GFP gene). A mega-nuclease (I-Sce I) site was introduced into the vector to facilitate transgene integration into zebrafish chromosomes. Zebrafish embryos were co-injected with iV7-GVP-UG and I-Sce I at one- or two-cell stage, raised to adults and checked for germ-line transmission of the transgene. Results: Transgenic lines bearing iV7-GVP-UG were established with relatively high transgenesis frequency (~20%). The transgenic F1 embryos exhibited GFP fluorescence in the limited numbers of cells in the eye, forebrain, hindbrain and spinal cord. The GFP distribution in the transgenic embryos was similar to the expression pattern of the endogenous VAL-opsin gene in wild-type embryos. Conclusions: The iV7-GVP-UG transgenic lines should be useful for future physiological experiments of VAL-opsin-expressing cells.
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