Transgenic/Molecular Approaches for Ocular Albinism

眼部白化病的转基因/分子方法

• 批准号: 6929014
• 负责人: DEBORA B FARBER
• 金额: $ 28.07万
• 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/6929014
• 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在发育中的视网膜和视觉通路内产生异常，导致永久性视力损害。虽然对与眼白化病（OA）和影响视网膜色素上皮（RPE）的相关色素减退病症相关的神经表型了解很多，但RPE内的这些变化如何影响神经系统仍然是个谜。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信号传导，G蛋白激活和最终调节神经视网膜基因表达的下游效应子之间关系的综合理解。将使用用于产生转基因小鼠的新的诱导位点特异性重组策略，其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，并将产生组成型活性的表达Gi的转基因小鼠，然后与Oa1敲除小鼠杂交，以观察Oa1敲除表型是否可以被挽救。与眼部色素减退相关的原发性神经异常是发育过程中视交叉处轴突导航的缺陷，表现为视轴突从颞部视网膜到大脑对侧的错误路由。将使用顺行和逆行追踪技术来定义这些各种转基因和基因敲除小鼠中与离视网膜途径相关的交叉模式，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。已经确定了在发展过程中的关键阶段，当RPE衍生的信号影响神经视网膜改变视交叉交叉的交叉模式，消减杂交策略结合微阵列分析将进行，以确定参与此信号的候选基因。将在Oa1敲除和Gi敲除小鼠中相对于野生型对照小鼠检查神经视网膜内和RPE细胞中基因表达的差异，然后将其与来自其中酪氨酸酶转基因被激活或保持失活的白化病小鼠的差异基因表达模式进行比较。使用这种结合发育生物学，分子遗传学和神经解剖学领域的方法，该研究计划将确定在RPE内启动并最终在视交叉处表现的关键信号事件。我们的研究应导致开发新的方法，用于设计基于基因治疗或Gi信号的药理学操作的治疗策略，以预防眼白化病和其他色素减退突变中的视觉障碍。