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Motokazu Tsujikawa, Takashi Nakao, Kohji Nishida; Rhodopsin S344x Transgenic Fish Cause Rapid Photoreceptor Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4354.
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© ARVO (1962-2015); The Authors (2016-present)
Rhodopsin is one of the important responsible genes for retinitis pigmentosa (RP). Several mutations in rhodopsin cause variable RP phenotypes. Among them, S344X causes severe phenotype. The purpose of this sturdy is to establish S344X mutant transgenic fish, and analyze their phenotype.
We cloned the whole length of human rhodopsin on the cloning vectors and introduced S344X 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 the several transgenic fish lines.
At 14 dpf, the number of rod photoreceptor in S344X was decreased under the half of that in wild-type transgenic (30.20 in S344X, 86.25 in wild-type). The difference is significant (p<0.01). TUNNEL assay also showed significant increase of apoptosis event in S344X retina. At 2 wpf, S344X retina showed 8.00 TUNEL positive cell at outer nuclear layer, whereas wt showed only 1.33 (p<0.01).
S334X mutation causes rapid photoreceptor degeneration. We conclude this transgenic fish mimics human RP and is useful as experimental model.
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