Genetics of Prion Susceptibility in vitro

体外朊病毒敏感性遗传学

• 批准号: 7298625
• 负责人: George A. Carlson
• 金额: $ 136.3万
• 依托单位: MC LAUGHLIN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7298625
• 关键词: Genetics; Prion; Susceptibility; vitro

Description (provided by applicant): The goals of this project are to dissect the biochemical networks that impinge on prion replication and to learn how prion replication causes neuronal dysfunction. The identification and characterization of genes in addition to Prnp that modify prion replication and susceptibility to prion disease has proven exceptionally difficult due to the need for incubation time studies in mice. Even the mechanisms by which alternative alleles of Prnp determine scrapie incubation time are unresolved. CNS stem cell neurosphere cultures can be produced from any strain or stock of mice, as well as from other species, and can be infected with prions. Neurosphere cultures provide a new approach to study prion biology and will be developed as a sensitive bioassay. Efficiency of infection, spread from cell to cell, and rate of prion replication can be discriminated in neurosphere cultures. Conformation dependent epitopes allow identification of individual cells with intracellular PrPSc; quantitative assays for infection, spread, and rate of replication will be refined. These parameters will be compared in prion strain-mouse strain combinations that vary in prion susceptibility or incubation time. Neurospheres that recapitulate the susceptibility of the mice of origin will form the substrates for dissection of the mechanisms and genes that are involved. Based on the hypothesis that alternative alleles of many quantitative trait loci reflect either differences in level or expression or different affinities for interacting molecules, RNAi will be used to evaluate effects of candidate genes on infection and production of PrPSc. The ability to produce neurosphere lines from any strain of mice permits testing modifiers using the same genetic background on which they were detected. CNS stem cells can be induced to differentiate along several pathways. The cell types produced under specific conditions from infected and non-infected neurosphere cultures will be compared, as will cell survival. Brain homogenates from mice infected with the RML prion strain diluted 10-8 can infect neurospheres from transgenic mice overexpressing PrP, and the limits of sensitivity have not yet been reached. Shortening the assay time from weeks to days will employ antibody epitopes present on PrP of the neurospheres, but not in PrPSc in the inoculum. This genetically tractable cell culture system has the potential to revolutionize prion research.

描述(申请人提供)：本项目的目标是剖析影响普恩复制的生化网络，并了解普恩复制如何导致神经元功能障碍。由于需要在小鼠中进行孵化时间研究，因此除了PRNP之外，识别和表征改变PrNP复制和对Prion疾病易感性的基因被证明是异常困难的。甚至PRNP的替代等位基因决定瘙痒病潜伏时间的机制也没有解决。中枢神经系统干细胞神经球培养物可以从任何品系或种群的小鼠身上产生，也可以从其他物种中产生，并可以被普恩病毒感染。神经球培养为研究蛋白生物学提供了一种新的方法，并将发展成为一种灵敏的生物测定方法。在神经球培养中，可以区分感染效率、细胞间传播和Pron复制率。构象依赖表位允许识别单个细胞与细胞内PrPSc；感染、传播和复制率的定量分析将得到改进。这些参数将在不同的Pron敏感度或孵化时间的Pron品系-小鼠品系组合中进行比较。神经球概括了原始小鼠的易感性，将形成剖析相关机制和基因的底物。基于多个数量性状基因座的替代等位基因反映水平或表达水平的差异或相互作用分子的不同亲和力的假设，RNAi将用于评估候选基因对PrPSc感染和产生的影响。从任何品系的小鼠产生脑球线的能力允许使用检测到修饰物的相同遗传背景来测试修饰物。中枢神经系统干细胞可以被诱导沿着几条途径分化。将比较感染和未感染的神经球培养物在特定条件下产生的细胞类型，以及细胞存活率。被稀释为10-8的RML PrP株感染的小鼠的脑匀浆可以感染过量表达PrP的转基因小鼠的神经球，但敏感性尚未达到极限。将检测时间从几周缩短到几天将利用神经球PrP上存在的抗体表位，而不是疫苗中PrPSc中的抗体表位。这种易于遗传处理的细胞培养系统有可能彻底改变普里恩的研究。