Transgenic/Molecular Approaches for Ocular Albinism

描述(申请人提供)：改变眼睛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，导致永久性视力损害。虽然与眼白化病(OA)相关的神经表型以及相关的影响视网膜色素上皮(RPE)的色素减少的条件已经知道了很多，但RPE内的这些变化如何影响神经系统仍然是一个谜。这项研究计划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中基因表达的下游效应器之间的关系的综合理解。将使用新的可诱导的位点特异性重组策略来产生转基因小鼠，这些策略允许在发育和控制转基因剂量的不同时间对所需基因进行组织特异性表达。还将检测三种不同的Gi蛋白基因敲除小鼠，以确定OA1正常发挥作用的Gi蛋白，并将产生具有结构性活性的表达Gi的转基因小鼠，然后与OA1基因敲除小鼠杂交，看看OA1基因敲除表型是否可以挽救。与眼部色素减退相关的原发神经异常是发育过程中视交叉的轴突导航缺陷，表现为视轴突从颞侧视网膜错误地进入大脑的对侧。在这些不同的转基因和基因敲除小鼠中，与视黄醇途径相关的交叉模式将使用顺行和逆行追踪技术来定义，而与其RPE相关的各种特征将被量化，包括酪氨酸酶表达的程度、总黑素含量和黑素小体的形态。在确定了发育过程中RPE衍生信号影响神经视网膜改变视交叉交叉模式的关键阶段后，将采用消减杂交策略与微阵列分析相结合的方法来识别参与这一信号传递的候选基因。将检测OA1基因敲除和GI基因敲除小鼠与野生型对照小鼠在神经视网膜和RPE细胞中基因表达的差异，然后与白化小鼠酪氨酸酶转基因被激活或保持不活跃的差异基因表达模式进行比较。利用发育生物学、分子遗传学和神经解剖学领域的这一组合方法，该研究计划将确定在RPE内启动并最终在视交叉表现的关键信号事件。我们的研究应该开发新的方法来设计基于GI信号的基因治疗或药物操作的治疗策略，以防止眼白化病和其他色素减退突变的视觉损害。

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.