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，隐孢子虫的能力增长了500倍。但是，仍然存在与在体外培养这种寄生虫有关的多个基本问题。这些问题之一是目前正在使用的培养基制剂的多样性，其中大多数是在体外种植寄生虫的最佳选择。导致最广泛的寄生虫发展的配方均与无性阶段和性阶段均取决于研究人员5-6关键补充剂，许多实验室未能进行修改，从而导致次优数据。这些问题中的第二个，更重要的是，我们无法在体外产生大量可行的卵囊。实验室必须使用脊椎动物来生产卵囊或依靠非常昂贵的商业来源。即使不包括费用，卵囊也必须从粪便中纯化，在此期间，它们通常会暴露于各种化学损伤中，包括二色剂和乙醚等试剂。另外，通常在添加10％（v/v）商业漂白剂（表面对卵囊进行消毒的10％（v/v））中，通常会消除微生物污染。因此，我们可以在体外获得足够数量的无需动物传播或大肠菌污染的任何方法都会打开各种目前不可用的途径。这些途径包括1）为更广泛的实验室提供机会，以减少的费用在寄生虫上工作，2）开发基因敲除和可以无限期地传播的等位基因替代品，3）3）进行代谢标记研究，并寻求将标签掺入纯净的ocyts，以及4）探索perforty的perforty confory controy controy的范围，并寻求将标签纳入perforty，并在4个）上进行了研究。事先暴露于溶剂或试剂的卵囊。该R21提案将尝试解决以上概述的两个问题：1）找到一种市售的，人造的，无血清的培养基，可在体外允许良好的寄生虫生长； 2）开发一个系统，我们可以在不需要实验动物的情况下定期，轻松，清洁和廉价地在体外产生足够数量的卵囊。