Sexual development of Toxoplasma in feline intestinal organoids and cell culture

 描述(申请人提供)：弓形虫的性周期仅限于猫的肠道，在那里形成数百万个卵囊，然后从它们的粪便中排出。这项建议的目的是重述组织培养中弓形虫的有性周期。为了实现这一目标，我们已经制定了一套方案来采集和培养极化的猫肠道上皮细胞。我们还已经改造了弓形虫寄生虫，使其表达一种来自无性阶段特定启动子的负性可选择标记，称为速殖子。添加负选择将抑制快速复制的速殖子裂解 肠道细胞，这将让有性阶段有时间发育。对于这个R21，我们将在我们极化的猫肠道附属物细胞中培养这些基因操纵的寄生虫，同时开发配子、二倍体形成和减数分裂的新标记，以确定性别进展的程度。我们R21的另一个目标是确定哪些永生的猫细胞最能概括性发育，这样它们就可以成为弓形虫群落的资源。我们已经用SV-40T抗原使我们的猫肠道上皮细胞永生化，我们将检测它们支持弓形虫性别发育的能力。我们还开始与密歇根大学的杰森·斯宾塞合作，在组织培养中创造猫肠道有机物质。创造体外的性发育条件将允许 生物过程的特定基因和待快速分析的冗余路径。它还将允许对弓形虫等原生寄生虫的完整生命周期进行分子分析。 无性发育已经可以在组织培养中进行。

 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 proposal is to recapitulate the T. gondii sexual cycle in tissue culture. Towards this goal, we have already developed a protocol to harvest and culture polarized feline intestinal epithelial cells. We have also already engineered T. gondii parasites to express a negative selectable marker from a promoter specific for the asexual stage called tachyzoite. Addition of the negative selection will inhibit the rapidly replicating tachyzoites from lysing the intestinal cells, which will give the sexual stages time to develop. For this R21, we will culture these genetically manipulated parasites in our polarized feline intestinal epithetical cells as wel as develop new markers for gametes, diploid formation and meiosis to determine the extent of sexual progress. Our other goal for this R21 is to define which immortal cat cells best recapitulate sexual development so that they can be a resource for the T. gondii community. We have already immortalized our feline intestinal epithelial cells with SV-40 T-antigen and we will examine their ability to support T. gondii sexual development. We have also begun a collaboration with Jason Spence at the University of Michigan to create cat intestinal organoids in tissue culture. Creation of ex vivo sexual development conditions will allow the contribution of specific genes to biological processes and redundant pathways to be rapidly analyzed. It will also allow for a molecular analysis of the complete lifecycle of a protistan parasite, as T. gondii asexual development can already be performed in tissue culture.