The use of PrP transgenic Drosophila to replace and reduce mice in the bioassay of mammalian prions

使用PrP转基因果蝇替代和减少小鼠在哺乳动物朊病毒的生物测定中的应用

• 批准号: NC/R00093X/1
• 负责人: Raymond Bujdoso
• 金额: $ 9.63万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FR00093X%2F1
• 关键词: use; PrP; transgenic; Drosophila; replace

Prion diseases include BSE of cattle, chronic wasting disease of cervids, scrapie of sheep and CJD of humans. These conditions are infectious and can spread between individuals of the same or different species. Animal prion diseases can be transmitted to humans and are therefore a threat to public health, evidenced by the outbreak of classical BSE in cattle followed by the emergence of variant CJD (vCJD) in humans. The pathogen that causes these transmissible diseases is an aggregated rogue form of a normal protein found in neurons and is referred to as a prion. Transmission of BSE to humans is believed to have occurred by dietary intake of BSE-contaminated food. Strict controls now protect humans from BSE, including removal of cattle tissues most likely to contain infectious prions when the animal is slaughtered. These cattle tissues are called specified risk material (SRM) and do not enter the human food chain.The only reliable way to detect BSE prion infectivity is by bioassay in experimental animals, which have traditionally been rodents, such as mice. The mouse prion bioassay involves injecting suspected BSE-infected samples into experimental mice and waiting to see if these animals develop prion disease. These assays are slow and cumbersome since the incubation time for prion disease in mice may be 1-2 years before clinical signs become evident and a predetermined end-point is reached. Collectively, BSE prion infectivity studies have utilised large numbers of mice over a long time period, and have subjected a high proportion of these animals to experience terminal clinical signs of experimental neurological disease. Since the emergence of classical BSE, the increased surveillance for the condition has led to identification of novel forms of the disease, which are a new threat to the human food chain as we do not know which tissues in affected cattle contain these new forms of BSE prion infectivity. It is essential therefore, to verify if the current SRM control measures are sufficient to prevent the new forms of BSE prions from entering the human food chain. Many more mice will be used in proposed food safety research programmes in order to measure prion infectivity levels in an extensive range of samples from cattle infected with new forms of BSE. In their entirety, these mouse prion bioassays will use more than 100,000 mice. It is vital to apply the 3Rs principles to this intended research programme by reducing and replacing, where possible, this large number of experimental mice with a prion bioassay that uses a less sentient host.In our laboratory we model prion disease in the fruit fly Drosophila melanogaster because they are relatively easy and economical to work with, and are a widely accepted ethical alternative to higher organisms including mice. We have developed a Drosophila-based prion bioassay that can detect BSE prion infectivity in order to provide an alternative to mice for the bioassay of bovine prions, and reduce the use of mice, and other vertebrate animal species, to measure prion infectivity in general. To do so, we have introduced a gene into the flies that allows them to produce the protein that aggregates in the brain of animals with prion disease. We already know that this mammalian protein will aggregate and cause prion disease in transgenic Drosophila when flies are fed infectious prions. What is equally significant is that the response by the transgenic flies to prion-infected material is evident within a few weeks following exposure to prion material. We can now develop a faster, more versatile and more sensitive bioassay to detect BSE prion infectivity than currently exists. We aim to transfer this know-how to the APHA, a main user and internationally recognised reference laboratory of the mouse prion bioassay. Our Skills and Knowledge Transfer to the APHA will provide the best opportunity to ensure world-wide impact of our new Drosophila-based prion bioassay.

朊病毒病包括牛的疯牛病、鹿的慢性消耗病、羊瘙痒病和人类的克雅氏病。这些疾病具有传染性，可以在相同或不同物种的个体之间传播。动物朊病毒疾病可以传播给人类，因此对公共卫生构成威胁，牛中爆发经典BSE，随后在人类中出现变异型CJD（vCJD）就是证明。导致这些传染性疾病的病原体是一种聚集的流氓形式的正常蛋白质，发现在神经元中，并被称为朊病毒。据信，疯牛病传播给人类是通过饮食摄入受疯牛病污染的食物发生的。现在，严格的控制措施保护人类免受疯牛病的侵害，包括在屠宰牛时去除最有可能含有传染性朊病毒的牛组织。这些牛组织被称为特定风险物质（SRM），不会进入人类食物链。检测疯牛病朊病毒感染性的唯一可靠方法是在实验动物中进行生物测定，传统上是啮齿动物，如小鼠。小鼠朊病毒生物测定包括将疑似BSE感染的样品注射到实验小鼠中，并等待观察这些动物是否发生朊病毒疾病。这些测定缓慢且繁琐，因为在临床症状变得明显并达到预定终点之前，小鼠中朊病毒疾病的孵育时间可能为1-2年。总的来说，BSE朊病毒感染性研究在很长一段时间内使用了大量小鼠，并且这些动物中有很高比例经历了实验性神经系统疾病的终末临床体征。自经典BSE出现以来，对该疾病的监测增加，导致发现了该疾病的新形式，这是对人类食物链的新威胁，因为我们不知道受影响牛的哪些组织含有这些新形式的BSE朊病毒感染性。因此，有必要验证目前的SRM控制措施是否足以防止新形式的BSE朊病毒进入人类食物链。在拟议的食品安全研究计划中将使用更多的小鼠，以测量感染新型疯牛病的牛的广泛样本中朊病毒的感染水平。总的来说，这些小鼠朊病毒生物测定将使用超过10万只小鼠。这是至关重要的应用3Rs原则，这一预期的研究计划，减少和取代，在可能的情况下，这一大量的实验小鼠与朊病毒的生物测定，使用较不敏感的host.In我们的实验室，我们模型朊病毒疾病的果蝇黑腹果蝇，因为他们是相对容易和经济的工作，是一个广泛接受的道德替代高等生物体，包括小鼠。我们已经开发了一种基于果蝇的朊病毒生物测定法，可以检测BSE朊病毒的感染性，以提供一种替代小鼠的牛朊病毒的生物测定，并减少使用小鼠和其他脊椎动物物种，以测量朊病毒的感染性一般。为了做到这一点，我们在果蝇中引入了一种基因，使它们能够产生蛋白质，这种蛋白质聚集在患有朊病毒疾病的动物的大脑中。我们已经知道，当果蝇被喂食传染性朊病毒时，这种哺乳动物蛋白会聚集并导致转基因果蝇患朊病毒病。同样重要的是，转基因果蝇对朊病毒感染物质的反应在暴露于朊病毒物质后的几周内是明显的。我们现在可以开发一种比现有的更快、更通用、更灵敏的生物测定法来检测BSE朊病毒的感染性。我们的目标是将这一技术转让给APHA，APHA是小鼠朊病毒生物测定的主要用户和国际公认的参考实验室。我们的技能和知识转移到APHA将提供最好的机会，以确保我们的新的基于果蝇的朊病毒生物测定的全球影响。