Identification of genetic modifiers of mammalian prion disease modelled in Drosophila

PHD项目战略主题：生物科学促进对健康的综合理解该项目的目的是确定在果蝇和小鼠身上模拟的获得性和遗传性哺乳动物Pron疾病的遗传修饰物。这将通过RNAseq分析从普恩病毒感染的神经元(包括总神经元和特定亚群)中分离出来的活跃翻译的mRNA来实现，以确定普恩病毒病的潜在遗传修饰物。在PrP转基因果蝇中，候选的遗传修饰物将通过调节它们的表达(分别通过RNAi或转基因过表达而减少或增加)来验证，并分析PrP转基因果蝇在PrP诱导的病理学中的任何变化。该项目的具体目标是：1.建立PrP/ribo-tag转基因果蝇Fly系，这些细胞系是哺乳动物PrP转基因的，并共表达一个核糖体标签(ribo-tag)，允许分离正在翻译的mRNA。Ribo-Tag果蝇将被转基因野生型绵羊或小鼠PrP，或小鼠PrP，其突变与人类遗传性Prion疾病(遗传性CJD)相关。核糖体标签和PrP都将在神经细胞或在特定的神经元亚群中表达。对PrP/Ribo-Tag转基因果蝇进行PrP感染的RNAseq分析分别在野生型绵羊和小鼠PrP转基因果蝇中建立获得性PrP病模型，分别接种绵羊和小鼠PrP转基因果蝇，在神经细胞或特定神经元亚群中表达PrP PAN。这将允许比较不同物种的PrP引起的神经病理，以及不同神经元亚群经历的神经退行性变之间的比较。将在转基因小鼠CJD PrP的果蝇中建立遗传性Pron病，以便识别获得性和遗传性PrP疾病的神经病理差异。与核糖体相关的具有核糖体标签的mRNA将从感染病毒的果蝇神经元中提取出来，并进行RNAseq。随后的生物信息学分析将确定生化途径，以及与这些途径相关的重要遗传修饰物，这些途径与不同果蝇模型中的获得性和遗传性Pron疾病有关。3.对Prion病PrP/Ribo-Tag转基因小鼠脑组织进行RNAseq分析从PrP/Ribo-Tag转基因小鼠脑中提取核糖体相关基因，进行RNAseq分析和生物信息学分析。这将确定与小鼠获得性Pron疾病有关的生化途径和遗传修饰物。4.遗传修饰物在果蝇Pron病中的调节表达将比较果蝇和小鼠RNAseq分析的数据，以确定这两种宿主共同的Prion病的遗传修饰物，从而验证果蝇模型的相关性。在PrP转基因果蝇中，PrP转基因果蝇在持续感染Prion期间，通过RNAi或转基因过表达来调节它们的表达，从而验证已确定的Prion疾病候选遗传修饰物的作用。基因修饰剂的有效性将通过评估PrP转基因果蝇PrP基因感染的PrP果蝇中PrP基因感染的PrP果蝇Prion种子活性的积累和Prion诱导的神经毒性表型(在存活和运动能力方面)的变化来确定。

PhD project strategic theme: Biosciences for an integrated understanding of healthThe aim of this project is to identify genetic modifiers of acquired and genetic mammalian prion disease modelled in Drosophila and mice. This will be achieved by RNASeq analysis of actively translated mRNA isolated from prion-infected neurons (both total neurons and specific subsets) in the brains of prion-diseased transgenic Drosophila and mice in order to identify potential genetic modifiers of prion disease. Candidate genetic modifiers will be validated through modulation of their expression (decreased or increased by RNAi or transgene over-expression, respectively) during ongoing prion disease in PrP transgenic Drosophila and analysing any changes in prion-induced pathology. The specific objectives of the project are:1. Generation of PrP / ribo-tag transgenic DrosophilaFly lines will be generated that are transgenic for mammalian PrP and co-express a ribosomal tag (ribo-tag) that allows isolation of mRNA undergoing translation. Ribo-tag Drosophila will be made transgenic for wild type ovine or murine PrP, or murine PrP with a mutation that correlates with genetic prion disease in humans (genetic CJD). Both the ribo-tag and the PrP will either be expressed pan neuronally or in specific neuronal subsets.2. Perform RNASeq analysis on prion-diseased PrP / ribo-tag transgenic DrosophilaAcquired prion disease will be established in wild type ovine or murine PrP transgenic Drosophila exposed to ovine or murine prion inoculum, respectively, that express PrP pan neuronally or in specific neuronal subsets. This will allow comparisons to be made between the neuropathology caused by different species' PrP, as well as comparisons between the neurodegeneration undergone by different neuronal subsets. Genetic prion disease will be established in Drosophila transgenic for murine-CJD PrP to allow differences to be identified between the neuropathology of acquired and genetic prion diseases. mRNA associated with ribosomes which possess the ribo-tag will be extracted from neurones of prion-diseased Drosophila and subjected to RNASeq. Subsequent bio-informatic analysis will identify biochemical pathways, along with important genetic modifiers associated with those pathways, that are involved in acquired and genetic prion disease in the different Drosophila models. 3. Perform RNASeq analysis on prion-diseased PrP / ribo-tag transgenic mouse brainsRibosomal-associated mRNA will be extracted from the brains of prion-diseased mice and subjected to RNASeq, and subsequent bio-informatic analysis. This will identify biochemical pathways and genetic modifiers involved in acquired prion disease in the mouse. 4. Modulated expression of genetic modifiers during prion disease in DrosophilaData from the Drosophila- and mouse-based RNASeq analyses will be compared to identify genetic modifiers of prion disease common between these two hosts, in order to validate the relevance of the Drosophila models. The role of identified candidate genetic modifiers of prion disease will be validated by modulation of their expression, achieved by RNAi or transgene over-expression, during an ongoing prion infection in PrP transgenic Drosophila. Validation of the genetic modifiers will be determined by assessing changes in the accumulation of prion seeding activity and the prion-induced neurotoxic phenotype (in terms of survival and locomotor ability) of prion-infected PrP transgenic Drosophila.