In Vitro Dissection of Genetic Susceptibility to Prion Disease

这个项目的目标是剖析影响Pron复制的生化网络，并 了解Prion复制如何导致神经元功能障碍。黄曲霉毒素相关基因的鉴定与鉴定 添加PRNP可以改变病毒复制和对病毒病的易感性已经被证明是例外的 由于需要在小鼠身上进行孵化时间研究，因此很困难。甚至是替代方案的机制 PRNP的等位基因决定了瘙痒病的潜伏期是未知的。中枢神经干细胞神经球培养可以 可由任何品系或种群的小鼠以及其他物种产生，并可被普恩病毒感染。 神经球培养为研究蛋白生物学提供了一种新的方法，并将被开发为一种敏感的 生物化验。可以区分感染效率、细胞间传播和Pron复制率 神经球培养。构象依赖表位允许识别单个细胞 细胞内PrPSc；感染、传播和复制率的定量分析将得到改进。这些 这些参数将在不同的Pron敏感度和不同的Pron敏感度或不同的鼠系组合中进行比较。 孵化时间。概括起源小鼠的易感性的神经球将形成 用于剖析所涉及的机制和基因的底物。基于这样的假设 许多数量性状座位的替代等位基因要么反映水平或表达的差异，要么反映不同的 相互作用分子的亲和力，RNAi将用于评估候选基因对感染和 PrPSc的生产。从任何品系的小鼠产生脑球线的能力允许进行测试 修饰剂使用与它们被检测到的相同的遗传背景。中枢神经系统干细胞可以被诱导 沿着几条路径进行区分。在特定条件下由感染和感染产生的细胞类型 将比较未感染的神经球培养物，以及细胞存活率。小鼠脑匀浆 感染稀释为10-8的RML病毒株可感染过量表达的转基因小鼠的神经球 PRP，敏感性尚未达到极限。将化验时间从几周缩短到几天 将使用神经球PrP上存在的抗体表位，而不是接种中PrPSc中的抗体表位。这 易于遗传处理的细胞培养系统有可能彻底改变普恩病毒的研究。

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 backgroundon 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.