Transgenic/Molecular Approaches for Ocular Albinism

描述（由申请人提供）：改变眼部色素沉着的基因突变会在发育中的视网膜和视觉通路中产生异常，导致永久性视力障碍。虽然我们对眼白化病（OA）相关的神经表型和影响视网膜色素上皮（RPE）的相关色素沉着状况了解很多，但RPE内的这些变化如何影响神经系统仍然是一个谜。该研究项目将直接解决这些问题，寻求对酪氨酸酶、黑色素合成、OA1信号、g蛋白激活和最终调节神经视网膜基因表达的下游效应物之间关系的综合理解。将采用新的可诱导位点特异性重组策略来产生转基因小鼠，从而允许在发育过程中的不同时间组织特异性表达所需基因和控制转基因剂量。我们还将检测三种不同的Gi蛋白敲除小鼠，以确定OA1正常发挥作用的Gi蛋白，并产生具有组成活性的表达Gi的转基因小鼠，然后与OA1敲除小鼠杂交，以观察是否可以挽救OA1敲除表型。与眼部色素沉着减退相关的原发性神经异常是发育过程中视交叉轴突导航的缺陷，表现为视轴突从颞视网膜到大脑另一侧的错误路线。在这些不同的转基因和基因敲除小鼠中，与视网膜通路相关的讨论模式将使用顺行和逆行通道追踪技术进行定义，而与它们的RPE相关的各种特征将被量化，包括酪氨酸酶表达程度、总黑色素含量和黑素体形态。在确定了rpe衍生信号影响神经视网膜改变视交叉讨论模式的关键发育阶段后，将采用结合微阵列分析的减法杂交策略来确定参与该信号的候选基因。与野生型对照小鼠相比，oa1基因敲除小鼠和gi基因敲除小鼠在神经视网膜和RPE细胞中基因表达的差异将被检测，然后与酪氨酸酶转基因被激活或保持失活的白化小鼠的差异基因表达模式进行比较。结合发育生物学、分子遗传学和神经解剖学等领域的研究方法，本研究项目将确定在RPE内启动并最终在视交叉处表现出来的关键信号事件。我们的研究将导致新的方法的发展，以设计基于基因治疗或Gi信号的药理操作的治疗策略，以防止眼白化病和其他低色素突变的视力损害。

DESCRIPTION (provided by applicant): Genetic mutations that alter ocular pigmentation produce abnormalities within the developing retina and visual pathways that cause permanent visual impairment. While much is known about the neural phenotype associated with ocular albinism (OA) and related hypopigmentation conditions affecting the retinal pigment epithelium (RPE), how these changes within the RPE affect the nervous system remain an enigma. This research program will directly address these issues, seeking an integrated understanding of the relationship between tyrosinase, melanin synthesis, OA1 signaling, G-protein activation and the downstream effectors that ultimately modulate gene expression in the neural retina. Novel inducible site-specific recombination strategies for generating transgenic mice will be used that permit tissue-specific expression of desired genes at different times during development and control of transgene dosage. Three different Gi protein knockout mice will also be examined to define the Gi protein through which OA1 normally functions, and constitutively active Gi-expressing transgenic mice will be generated and then crossed to Oa1-knockout mice to see whether the Oa1-knockout phenotype can be rescued. The primary neural abnormality associated with ocular hypopigmentation is a defect in axonal navigation at the optic chiasm during development, manifested as a misrouting of optic axons from the temporal retina into the opposite side of the brain. The decussation patterns associated with the retinofugal pathways in these various transgenic and knockout mice will be defined using anterograde and retrograde tract-tracing techniques, while various features associated with their RPE will be quantified, including the degree of tyrosinase expression, total melanin content and melanosomal morphology. Having identified the critical stages during development when an RPE-derived signal affects the neural retina altering decussation patterns at the optic chiasm, a subtractive hybridization strategy combined with microarray analysis will be conducted to identify candidate genes involved in this signaling. Differences in gene expression within the neural retina and in RPE cells will be examined in Oa1-knockout and Gi-knockout mice relative to wild-type control mice, and then compared with patterns of differential gene expression derived from albino mice in which a tyrosinase transgene is activated or remains inactive. Using this combination of approaches drawing on the fields of developmental biology, molecular genetics and neuroanatomy, this research program will identify the critical signaling events initiated within the RPE and ultimately manifested at the optic chiasm. Our studies should lead to the development of new approaches for devising therapeutic strategies based on gene therapy or pharmacological manipulations of Gi signaling in order to prevent the visual impairments in ocular albinism and other hypopigmentation mutations.