In Vitro Dissection of Genetic Susceptibility to Prion Disease

朊病毒病遗传易感性的体外剖析

• 批准号: 7299474
• 负责人: George A. Carlson
• 金额: $ 45.69万
• 依托单位: MC LAUGHLIN RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7299474
• 关键词: Affinity; Alleles; Antibodies; Biochemical; Biological Assay; Biological Markers; Biology; Bos taurus; Brain; Candidate Disease Gene; Cattle; Cell Culture System; Cell Differentiation process; Cell Survival; Cells; Classification; Collaborations; Condition; Dissection; Epitopes; Gene-Modified; Genes; Genetic; Genetic Models; Genetic Predisposition to Disease; Genome; Goals; Human; In Vitro; Individual; Infection; Learning; Molecular Conformation; Mouse Strains; Mus; Mutation; Neuronal Dysfunction; Numbers; Pathway interactions; PrPSc Proteins; Predisposition; Prion Diseases; Prions; Production; Protein Overexpression; Quantitative Trait Loci; RNA Interference; Rate; Research; Scrapie; Stem cells; Testing; Time; Time Study; Transgenic Mice; Week; Work; base; cell type; cervid; day; in vitro Bioassay; middle lung lobe; novel; novel strategies

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.

这个项目的目标是剖析影响朊病毒复制的生化网络