Generation and Characterization of a Novel Cell Subpopulation Ablation Model

项目概要/摘要：本申请的主要目标是，第一次， 表征用于消融细胞亚群的创新资源。此资源将有助于学习角色 不同亚型的细胞及其在发病机制、组织修复和体内再生中的相互作用。我们 我建议通过产生一种针对细胞亚群的新工具来提高细胞消融的资源 由双重表型标记定义的群体。使用条件性靶向的功能丧失研究 细胞消融已被广泛用于研究细胞功能和相互作用、组织修复和分化 尽管它们仅靶向一种标记物标记的细胞群。先进的方法正在改变， 加快我们识别和理解免疫、神经和其他细胞亚群的方法。这些 细胞亚群可以用多种表型标记物标记。我们还预计， 免疫或神经亚群的谱将在大脑倡议的时代和 后人类基因组计划时代。虽然目前的工具可用于消融细胞群，但它们 不能特异性靶向细胞亚群，因为它们只能靶向一种标记，而不能靶向双标记细胞。 因此，需要开发一种仪器，其能够消除动物中的细胞亚群的损失- 功能研究。为此，我们建议产生中间溶素（ILY）介导的细胞亚群 烧蚀模型ILY是由中间链球菌（SI）分泌的一种毒素，仅与人细胞结合 膜蛋白CD 59（hCD 59），但不与任何其他物种的CD 59。一旦被束缚， 溶解细胞我们最近建立了Cre诱导的hCD 59转基因小鼠系（ihCD 59），其中hCD 59 表达仅发生在Cre介导的重组之后。向Cre+/-的各种细胞系施用ILY ihCD 59 +/-小鼠导致免疫、上皮或神经细胞的快速和特异性消融，而没有脱靶 方面的影响.我们还在几种疾病模型中测试了ILY/ihCD 59介导的细胞消融方法，以研究 免疫细胞功能、肝细胞和/或胆管上皮损伤和再生以及神经细胞损伤。 该细胞系（ihCD 59）仅靶向一种标记物标记的细胞群，不能用于消融亚群细胞 人口为了进一步推进这一工具对亚群细胞消融，我们建议开发和表征 CAG（CMV早期增强子/鸡β肌动蛋白）-STOP-STOP-STOP-Frt-STOP-Frt（DihCD 59）或双 诱导型小鼠品系，其中转基因（hCD 59）表达仅在Cre和Flp介导的 组合事件具体来说，我们将检验我们的工作假设，即1）hCD 59表达将是 在靶向亚群中通过Cre和Flp介导的组合事件特异性介导， DihCD 59或通过产生三重（Cre+/Flp+/DihCD 59+）转基因阳性小鼠;和2）ILY注射至 三重转基因阳性小鼠将切除细胞亚群以进一步研究它们的体内功能。

Project Summary/Abstract: The primary objective of this application is, for the first time, to generate and characterize an innovative resource for ablating subsets of cells. This resource will facilitate studying the roles of different subtypes of cells and their interactions in pathogenesis, tissue repair, and regeneration in vivo. We propose to advance the resources of cell ablation by generating a novel tool for targeting the subsets of cell populations that are defined by dual phenotypical markers. Loss-of-function studies using conditional targeted cell ablation have been widely used to investigate cell function and interaction, tissue repair and differentiation in vivo though they target only one marker-labelled cell population. Advanced methodologies are changing and expediting the ways in which we identify and understand immune, neural, and other cell subpopulations. These cell subpopulations can be labelled by the multiple phenotypical markers. We also anticipate that a growing spectrum of immune or neural subpopulations will be discovered in The Era of the Brain Initiative and The Post-Human Genome Project Era. Although current tools are available for ablating a cell population, they cannot specifically target cell subpopulations because they can only target one maker, not dual-labelled cells. Therefore, there is a need to develop an instrument that is able to eliminate subsets of cells in animals for loss- of-function studies. To this end, we propose to generate an intermedilysin (ILY)-mediated cell subpopulation ablation model. ILY, a toxin secreted by Streptococcus intermedius (SI), exclusively binds to the human cell membrane protein CD59 (hCD59), but not to CD59 of any other species. Once bound, ILY rapidly and potently lyses the cells. We recently established a Cre-inducible hCD59 transgenic mouse line (ihCD59) where hCD59 expression only occurs after Cre-mediated recombination. Administration of ILY to various lines of Cre+/- ihCD59+/- mice resulted in rapid and specific ablation of immune, epithelial or neural cells without off-target effects. We also tested the ILY/ihCD59-mediated cell ablation method in several disease models to study immune cell functions, hepatocyte and/or biliary epithelial damage and regeneration, and neural cell damage. This line (ihCD59) targets only one marker-labelled cell population, and cannot be used for ablating subset cell population. To further advance this tool towards subset cell ablation, we propose to develop and characterize CAG (CMV early enhancer/chicken beta actin)-floxP-STOP-floxP-Frt-STOP-Frt (DihCD59) or a double inducible mouse strain where the transgene (hCD59) expression occurs only following Cre- and Flp-mediated combinational events. Specifically, we will examine our working hypotheses that 1) hCD59 expression will be specifically mediated in the targeted subpopulations by Cre- and Flp-mediated combinational events in DihCD59 or by generating triple (Cre+/Flp+/DihCD59+) transgene positive mice; and that 2) ILY injection to the triple transgene positive mice will ablate subpopulations of cells for further investigating their functions in vivo.