Transgenic/Molecular Approaches for Ocular Albinism

眼部白化病的转基因/分子方法

• 批准号: 7084565
• 负责人: DEBORA B FARBER
• 金额: $ 27.31万
• 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/7084565
• 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）：改变眼色素沉着的基因突变会在发育中的视网膜和视觉途径中产生异常，从而导致永久视觉障碍。尽管对与视网膜色素上皮（RPE）相关的眼白化病（OA）（OA）（OA）（OA）和相关的不形成性条件相关的神经表型知之甚少，但RPE内的这些变化如何影响神经系统仍然是谜。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。将使用新的可诱导位点特异性重组策略来产生转基因小鼠，以便在转基因剂量的开发和控制过程中，在不同时间的组织特异性表达。还将检查三种不同的GI蛋白基因敲除小鼠，以定义OA1通常功能的GI蛋白，并且将生成组成型活性的表达转基因小鼠，然后将其交叉到OA1-KNOCKOUT小鼠以查看是否可以营救OA1-Knockout表型。与眼部异常相关的主要神经异常是发育过程中视线chiAsm处的轴突导航的缺陷，这表现为对颞视网膜视网膜视网膜的误射到大脑的另一侧。在这些各种转基因和基因敲除小鼠中，与视网膜途径相关的de脉模式将使用顺行和逆行追踪技术定义，而与其RPE相关的各种特征将被量化，包括酪氨酸酶表达，总黑色素含量和黑色素体形态。在确定了开发过程中的临界阶段，当RPE衍生的信号影响神经视网膜改变了视神经误差模式时，将进行减法杂交策略与微阵列分析相结合，以识别该信号中涉及的候选基因。相对于野生型对照小鼠，将在OA1-敲除和Gi-Knockout小鼠中检查基因表达和RPE细胞中的基因表达差异，然后将其与来自白化小鼠的差异基因表达的模式进行比较，在该鉴定基因表达的模式中，酪氨酸酶转移激活或保持活性或保持活性。通过在发育生物学，分子遗传学和神经解剖学领域的这种方法的组合，该研究计划将确定在RPE内引发的关键信号事件，并最终在视觉上表现出来。我们的研究应导致开发基于基因治疗或GI信号的药理学操纵来制定治疗策略的新方法，以防止眼睛白化病和其他低调突变的视觉障碍。