Genetics of Prion Susceptibility in vitro

体外朊病毒敏感性遗传学

• 批准号: 7644935
• 负责人: George A. Carlson
• 金额: $ 137.3万
• 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/7644935
• 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（由申请人提供）：该项目的目标是剖析影响朊病毒复制的生化网络，并了解朊病毒复制如何导致神经元功能障碍。由于需要在小鼠中进行孵育时间研究，除Prnp外，鉴定和表征改变朊病毒复制和对朊病毒疾病易感性的基因已被证明异常困难。甚至Prnp的替代等位基因决定痒病潜伏期的机制也没有得到解决。中枢神经系统干细胞神经球培养物可以从任何品系或小鼠种群中产生，也可以从其他物种中产生，并且可以被朊病毒感染。神经球培养为研究朊病毒生物学提供了一种新的途径，并将发展成为一种灵敏的生物测定方法。感染的效率，从细胞到细胞的传播，和朊病毒复制的速度可以在神经球培养中区分。构象依赖的表位允许鉴定细胞内PrPSc的单个细胞；对感染、传播和复制率的定量分析将得到改进。这些参数将在不同朊病毒易感性或孵育时间的朊病毒毒株-小鼠毒株组合中进行比较。再现原鼠易感性的神经球将成为解剖相关机制和基因的基础。基于许多数量性状位点的替代等位基因反映了相互作用分子的水平或表达差异或不同亲和力的假设，RNAi将用于评估候选基因对PrPSc感染和产生的影响。从任何小鼠品系中产生神经球系的能力允许使用与检测到它们相同的遗传背景来测试修饰物。中枢神经系统干细胞可以通过几种途径诱导分化。将比较在特定条件下从感染和未感染的神经球培养物中产生的细胞类型，以及细胞存活率。经稀释10-8的RML朊病毒株感染的小鼠脑匀浆可感染过表达PrP的转基因小鼠的神经球，敏感性尚未达到极限。将测定时间从数周缩短到数天，将利用存在于神经球PrP上的抗体表位，而不是接种物PrPSc上的抗体表位。这种基因易处理的细胞培养系统有可能彻底改变朊病毒的研究。