In vitro refinements for Cryptosporidium parvum

小隐孢子虫的体外改良

• 批准号: 6553262
• 负责人: Steve J Upton
• 金额: $ 18.19万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-15 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6553262
• 关键词: Cryptosporidium; chick embryo; cow; cryptosporidiosis; deficient growth media; egg /ovum; enzyme linked immunosorbent assay; gene targeting; laboratory mouse; microorganism reproduction; technology /technique development; tissue /cell culture

DESCRIPTION (provided by applicant): Over the last decade, our ability to cultivate the intestinal protozoan, Cryptosporidium parvum, in cell culture has increased by >500x. However, there still exist multiple fundamental problems associated with cultivating this parasite in vitro. One of these problems is the diversity of medium formulations currently in use, most of which are sub-optimal for growing the parasites in vitro. Formulations which result in the most extensive parasite development complete with both asexual and sexual stages rely on the investigatoradding5-6 key supplements and many laboratories fail to make the modifications resulting in sub-optimal data. The second of these problems, and more importantly, is our inability to produce high numbers of viable oocysts in vitro. Laboratories must either use vertebrate animals to produce oocysts or rely on commercial sources which are extremely expensive. Even excluding the expense, oocysts must be purified from feces at which time they are often exposed to a variety of chemical insults including reagents such as potassium dichromate and ether. In addition, microbial contamination is normally eliminated before the parasites are utilized by the addition of 10% (v/v) commercial bleach, which surface sterilizes the oocysts. Thus, any method where we can obtain sufficient numbers of oocysts in vitro without the need for animal propagation or coliform contamination would open up a variety of avenues that are currently unavailable. These avenues include1) providing opportunities to a wider variety of laboratories to work on the parasite at decreased expense, 2) developing gene knock-outs and allelic replacements that could be propagated indefinitely, 3) performing metabolic labeling studies and looking at incorporation of label into the purifiable oocysts, and 4) more easily studying parasite development in vitro following exposure of oocysts to disinfectants without pre-exposing oocysts to solvents or reagents beforehand. This R21 proposal will attempt to solve the two problems outlined above: 1) find a commercially available, artificial, serum-free medium that permits good parasite growth in vitro; and 2) develop a system whereby we can routinely, easily, cleanly, and inexpensively generate sufficient numbers of oocysts in vitro without the need for experimental animals.

描述（由申请人提供）：在过去的十年中，我们在细胞培养中培养肠道原生动物，小隐孢子虫的能力增加了1000倍。然而，体外培养这种寄生虫仍然存在许多基本问题。其中一个问题是目前使用的培养基配方的多样性，其中大多数都不是体外培养寄生虫的最佳选择。导致最广泛的寄生虫发育的配方，包括无性和有性阶段，依赖于研究人员添加5-6个关键补充剂，许多实验室未能进行修改，导致次优数据。第二个问题，也是更重要的问题，是我们无法在体外产生大量可存活的卵囊。实验室必须要么使用脊椎动物来生产卵囊，要么依靠极其昂贵的商业来源。即使不考虑费用，卵囊也必须从粪便中纯化，在此期间，卵囊经常暴露在各种化学物质中，包括重铬酸钾和乙醚等试剂。此外，通常在寄生虫被利用之前，通过添加10% （v/v）的商业漂白剂来消除微生物污染，这种漂白剂可以对卵囊进行表面消毒。因此，任何可以在体外获得足够数量的卵囊而不需要动物繁殖或大肠菌群污染的方法都将开辟目前无法获得的各种途径。这些途径包括：1)为更广泛的实验室提供以更低成本研究寄生虫的机会；2)开发可以无限繁殖的基因敲除和等位基因替代；3)进行代谢标记研究，并考虑将标记纳入可纯化的卵囊；4)更容易在体外研究卵囊暴露于消毒剂后的寄生虫发育，而无需事先将卵囊暴露于溶剂或试剂中。该R21提案将试图解决上述两个问题：1)找到一种市售的、人工的、无血清的培养基，使寄生虫在体外生长良好；2)开发一种系统，使我们能够常规、简单、清洁、廉价地在体外产生足够数量的卵囊，而不需要实验动物。