Transgenic/Molecular Approaches for Ocular Albinism

眼部白化病的转基因/分子方法

• 批准号: 6702942
• 负责人: DEBORA B FARBER
• 金额: $ 27.94万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6702942
• 关键词: G protein; albinism; albino mouse; cell component structure /function; developmental neurobiology; eye disorder; gene expression; gene targeting; genetically modified animals; melanins; melanosomes; microarray technology; monophenol monooxygenase; neural transmission; neuronal guidance; neuronal transport; optic chiasmas; optic tract; receptor coupling; retinal pigment epithelium; subtraction hybridization; visual pigments

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.

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，从而导致永久性视力障碍。虽然人们对与眼白化病 (OA) 相关的神经表型以及影响视网膜色素上皮 (RPE) 的相关色素沉着不足状况了解很多，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基因表达的下游效应器之间关系的综合理解。将使用新的可诱导位点特异性重组策略来产生转基因小鼠，该策略允许在发育和转基因剂量控制期间的不同时间组织特异性表达所需基因。还将检查三种不同的Gi蛋白敲除小鼠，以确定OA1正常发挥功能的Gi蛋白，并产生组成型活性表达Gi的转基因小鼠，然后与Oa1敲除小鼠杂交，看看是否可以挽救Oa1敲除表型。与眼部色素沉着不足相关的原发性神经异常是发育过程中视交叉处的轴突导航缺陷，表现为视轴突从颞部视网膜错误路由到大脑的另一侧。将使用顺行和逆行束追踪技术来定义与这些不同转基因和敲除小鼠中的视网膜离走通路相关的交叉模式，同时将量化与其RPE相关的各种特征，包括酪氨酸酶表达程度、总黑色素含量和黑素体形态。确定了发育过程中 RPE 衍生信号影响神经视网膜并改变视交叉交叉模式的关键阶段后，将采用消减杂交策略结合微阵列分析来识别参与该信号传导的候选基因。将检查 Oa1 敲除和 Gi 敲除小鼠相对于野生型对照小鼠神经视网膜和 RPE 细胞内基因表达的差异，然后与来自酪氨酸酶转基因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。该研究项目将利用发育生物学、分子遗传学和神经解剖学领域的方法组合，识别 RPE 内启动并最终在视交叉处表现出来的关键信号事件。我们的研究应该导致开发新方法，以设计基于基因治疗或 Gi 信号传导的药理学操作的治疗策略，以预防眼部白化病和其他色素沉着不足突变引起的视力障碍。