Generation and Characterization of a Novel Cell Subpopulation Ablation Model

新型细胞亚群消融模型的生成和表征

• 批准号: 9932578
• 负责人: Xuebin Qin
• 金额: $ 21.25万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-21 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9932578
• 关键词: Ablation; Animal Model; Animals; B-Lymphocytes; BRAIN initiative; Biliary; Binding; CD4 Positive T Lymphocytes; Cell Communication; Cell Membrane Proteins; Cell physiology; Cells; Chickens; Chronic; Cytomegalovirus; Disease; Disease model; Dose; Engineering; Enhancers; Epithelial; Epithelial Cells; Event; Experimental Autoimmune Encephalomyelitis; FOXP3 gene; Generations; Genetic Recombination; Genetic Transcription; Goals; Hepatocyte; Human; Human Genome Project; Immune; Immune response; Inflammatory; Injections; Interneurons; Label; LoxP-flanked allele; Mediating; Methodology; Methods; Modeling; Mouse Strains; Multiple Sclerosis; Mus; Natural regeneration; Neurons; Parvalbumins; Pathogenesis; Pharmacology; Population; Recovery; Regulatory T-Lymphocyte; Resources; Rodent; Role; Somatostatin; Streptococcus intermedius; T-Lymphocyte; T-Lymphocyte Subsets; Tars; Testing; Time; Tissue Differentiation; Tissues; Transgenes; Transgenic Mice; Transgenic Organisms; alpha Toxin; beta Actin; cell injury; cell type; diphtheria toxin receptor; in vivo; in vivo regeneration; innovation; instrument; loss of function; macrophage; novel; phenotypic biomarker; relating to nervous system; response; tissue regeneration; tissue repair; tool; transcription factor; γδ T cells

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.

项目摘要/摘要：本应用程序的主要目标是首次生成和 描述一种用于消融细胞亚群的创新资源。这一资源将有助于研究角色 不同亚型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 提出通过产生一种新的靶向细胞亚群的工具来推进细胞消融资源 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 然而，在体内，它们只针对一个标记的细胞群体。先进的方法正在发生变化， 加快我们识别和理解免疫、神经和其他细胞亚群的方式。这些 细胞亚群可以用多个表型标记来标记。我们还预计，越来越多的 免疫或神经亚群的光谱将在大脑倡议和 后人类基因组计划时代。尽管目前的工具可用于消融细胞群体，但它们 不能专门针对细胞亚群，因为它们只能针对一个标记，而不是双标记细胞。 因此，有必要开发一种能够消除动物细胞亚群丢失的仪器- 功能障碍研究。为此，我们建议产生一个中间溶素(ILY)介导的细胞亚群 消融模型。伊利，一种由中间链球菌(SI)分泌的毒素，专门与人类细胞结合 膜蛋白CD59(HCD59)，但不与其他物种的CD59结合。一旦绑定，ILY就会迅速而有力地 裂解细胞。我们最近建立了一个Cre诱导的hCD59转基因小鼠系(IhCD59)，其中hCD59 只有在Cre介导的重组之后才能表达。对CRE+/-多条线路的ILY管理 IhCD59+/-小鼠可快速、特异地消融免疫细胞、上皮细胞或神经细胞，而不会偏离靶点 效果。我们还在几种疾病模型中测试了ILY/ihCD59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。 这条线(IhCD59)只针对一个标记的细胞群体，不能用于消融子集细胞 人口。为了进一步将这一工具推向亚群细胞消融，我们建议开发和表征 CAG(CMV早期增强子/鸡β肌动蛋白)-FLOXP-STOP-FLOXP-FRT-STOP-FRT(DihCD59)或双 转基因(HCD59)仅在Cre和FLP介导下表达的可诱导小鼠品系 组合事件。具体地说，我们将检验我们的工作假设，即1)hCD59表达将是 Cre和FLP介导的结合事件在目标亚群中的特异性介导 DihCD59或通过产生三重(Cre+/FLP+/DihCD59+)转基因阳性小鼠；以及2)ILY注射到 三重转基因阳性小鼠将去除细胞亚群，以便进一步研究它们在体内的功能。