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Hannah Elizabeth Henson, Marie Ann Forbes-Osborne, Sara N Perkins, Ann C Morris, Retinal Cell Biology; PROGRESS TOWARDS GENERATING ZEBRAFISH MODELS OF RETINITIS PIGMENTOSA. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5389.
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© ARVO (1962-2015); The Authors (2016-present)
The inherited disease retinitis pigmentosa (RP) causes progressive degeneration of photoreceptors with a number of cases due to mutations in rhodopsin (RHO). Zebrafish provide an attractive model for retinal diseases because they possess a cone-rich retina. Here, we describe progress towards generating constitutive and inducible zebrafish lines expressing mutant RHO alleles previously identified in patients with RP, thereby allowing us to study the genotype-phenotype relationship in photoreceptor degeneration. Also, because zebrafish regenerate photoreceptors, we can discover pathways promoting retinal regeneration, which may suggest novel approaches to induce regeneration in mammals.
For constitutive lines, mutations in human RHO included P23H, R135L, and K296E. Using the Tol2 kit, mutant RHOs were subcloned from pGEM®-T into pME-MCS using pME-mCherry as a control. We also subcloned the Xenopus rhodopsin promoter (XOPS5.5) into the p5E-MCS vector. Using LR cloning, p5E(XOPS5.5), pME(RHOs or mCherry), and p3E(polyA) were inserted into the pDestTol2CG2 vector. To confirm constitutive constructs were functional, we injected one- to two-cell stage embryos with 30 pg of pDestTol2CG2(XOPS5.5:mCherry:polyA) DNA or 30pg DNA + 30 pg transposase RNA. Embryos were screened for mCherry expression in rods. Inducible lines were generated using the Tet-ON system and a biTRE-mCherry vector. Mutant RHOs were subcloned into pME-MCS. The p5E(dA-mCherry:biTRE), pME(RHOs), and p3E(polyA) were inserted into the pDestTol2CG2 vector.
We successfully generated constructs for P23H, R135L and K296E constitutive lines, along with mCherry. For embryos injected with pDestTol2CG2(XOPS5.5:mCherry:polyA) DNA, 41% survived past 3 dpf of which 32% had mCherry+ rods. For those co-injected with DNA + transposase, 33% survived past 3dpf and 20% of those had mCherry+ rods. For inducible lines, we successfully generated constructs for the P23H, R135L, and K296E mutations.
We confirmed that XOPS5.5 drives expression of mCherry in rods. We also generated constructs expressing mutations in human RHO and inducible constructs co-expressing mutant RHO and mCherry. Future experiments will generate stable transgenic lines expressing mutant RHOs. The inducible lines will enable us to induce expression at various developmental stages to study degeneration, and turn off expression to study retinal regeneration.
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