Sexual development of Toxoplasma in feline intestinal organoids and cell culture

 描述（由申请方提供）：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，随后通过粪便排泄。这个建议的目的是概括T。组织培养中的弓形虫性周期。为了实现这一目标，我们已经制定了一个协议，收获和培养极化猫肠上皮细胞。我们还设计了T。弓形虫表达来自称为速殖子的无性阶段特异性启动子的阴性选择标记。加入阴性选择将抑制快速复制的速殖子裂解细胞， 肠细胞，这将使性阶段的时间发展。对于这个R21，我们将培养这些基因操纵的寄生虫在我们的极化猫肠上皮细胞以及开发新的标记配子，二倍体形成和减数分裂，以确定性进展的程度。我们对R21的另一个目标是确定哪些永生猫细胞最能再现性发育，以便它们可以成为T细胞的资源。弓形虫群落我们已经用SV-40 T抗原使猫肠上皮细胞永生化，我们将检测它们支持T细胞的能力。弓形虫性发育我们还开始与密歇根大学的Jason Spence合作，在组织培养中创造猫肠道类器官。创造离体性发育条件将允许 特定基因的生物过程和冗余的途径进行快速分析。它还将允许对原生生物寄生虫的完整生命周期进行分子分析，如T。弓形虫 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.