Analysis of Toxoplasma sexual development in polarized cat intestinal cells

描述（由申请人提供）：刚地弓形虫的性循环仅限于猫的肠道，在那里形成数百万个卵囊并随后随粪便排出。该应用程序的目的是在组织培养中重现弓形虫的性循环。为了实现这一目标，我们已经制定了一套收集和培养极化猫肠上皮细胞的方案。我们对这种R03的目标是使这些猫肠上皮细胞永生，同时保持它们极化的能力。我们也已经设计了弓形虫寄生虫来表达来自无性阶段特异性启动子的负选择性标记。对于这种R03，我们将使用负选择方法在猫肠道上皮细胞中培养这些基因操纵的寄生虫，以抑制无性期的生长。这种抑制将阻止快速复制的形式，称为速殖子，从分解肠细胞。抑制宿主细胞裂解将给性阶段的发展时间。目前，刚地弓形虫的有性杂交数量有限，因为它们必须在感染后3-10天喂养被感染的老鼠大脑并收集其粪便后才能在活猫中进行。体外性发育条件的创造将使经典的遗传杂交在实验室中成为常规，这将开辟多种新的实验设计策略。它还将允许对原生动物寄生虫的完整生命周期进行分子分析，因为弓形虫的无性发育已经可以在组织培养中进行。组织培养中弓形虫性发育的决定性条件的另一个好处将是生产稳定的疫苗。作为卵囊生产的弓形虫疫苗将是理想的，因为它在任何环境条件下都是稳定的（无需冷藏），而且它是口服接种（无需针头）。我们已经非常成功地产生了必要的工具，开始研究弓形虫在组织培养中的性发育。我们现在将使用这些工具来选择在离体猫肠细胞中进行性发育的寄生虫。组织培养中弓形虫性循环的任何进展都将是该领域的重大进步，因为可以开始对这些顽固性阶段进行分子分析。

DESCRIPTION (provided by applicant): The sexual cycle of Toxoplasma gondii is limited to the feline intestine where millions of oocysts are formed and subsequently excreted in their feces. The goal of this application is to recapitulate the T. gondii sexual cycle in tissue culture Towards this goal, we have already developed a protocol for harvesting and culturing of polarized feline intestinal epithelial cells. Our goal for this R03 is to immortalize these cat intestinal epithelial cells while maintaining their ability to polarize. We have also already engineered T. gondii parasites to express a negative selectable marker from an asexual stage-specific promoter. For this R03 we will culture these genetically manipulated parasites in the feline intestinal epithetical cells using the negative selection to inhibit the growth of the asexul stages. This inhibition will prevent the rapidly replicating form, called tachyzoites, from lysing he intestinal cells. Inhibition of host cell lysis will give the sexual stages time to develop. Curretly, only a limited number of sexual crosses of T. gondii are performed because they must occur in live cats after feeding them infected mouse brains and collecting their feces 3-10 days after infection. Creation of ex vivo sexual development conditions will allow classical genetic crosses to become routine in laboratories, which will open multiple new experimental design strategies. It will also allow for a molecular analysis of the complete lifecycle of a protozoan parasite, as T gondii asexual development can already be performed in tissue culture. Another benefit of the determining conditions for T. gondii sexual development in tissue culture will be the production of a stable vaccine. A T. gondii vaccine produced as an oocyst will be ideal because it will be stable in any environmental conditions (no refrigeration) and it will be an oral inoculation (no needles). We have been highly successful generating the tools necessary to begin sexual development studies of T. gondii in tissue culture. We will now use these tools to select parasites undergoing sexual development in ex vivo feline intestinal cells. Any progression through the sexual cycle of T. gondii in tissue culture will be a major advance for the field because a molecular analysis of these recalcitrant stages can begin.