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Y. Sasamoto, T. Nakao, N. Matsumura, M. Tsujikawa; Generation of Transgenic Zebrafish Lines With Human Rhodopsin Mutations. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1354.
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© ARVO (1962-2015); The Authors (2016-present)
There are many rhodopsin mutations known to cause retinitis pigmentosa (RP). Interestingly, it is known that these mutations show different phenotypes maybe by different pathogenic mechanisms. To investigate these differences, we produced the series of transgenic zebrafish which possess such rhodopsin mutations.
We cloned the whole length of human rhodopsin on the cloning vectors and introduced various rhodopsin mutations into the vectors using PCR method. Then, we subcloned the vectors on our over expression vector, which contains zebrafish rhodopsin promoter to express the gene in rod photoreceptor and tol2 transposon site to facilitate the genome integration of the mutated gene. We injected the constructs into the fertilized eggs with tol2 mRNA to obtain G0 mosaic fish. After they grew up, we crossed these G0 injected fish and wt fish to obtain transgenic fish lines.
We had obtained transgenic zebrafish lines of Q344X and P23H which are popular in RP patients in the Western countries. We also made G106R, G188R, K296E, T4R, N15S and L88P mutant lines which are seen in Japan. Q344X transgenic fish showed the significant decrease of photoreceptor cells at 5 dpf. TUNEL assay revealed the decrease is due to photoreceptor cell death.
We have created a lot of rhodopsin mutant fish lines, which accelerates the understanding of pathogenesis of human RP. In addition, these zebrafish may be useful for evaluating novel therapies, which also lead to order-made treatment.
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