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：此应用程序的主要目的是首次生成和 表征用于消融细胞子集的创新资源。该资源将有助于研究角色 细胞的不同亚型及其在体内的发病机理，组织修复和再生中的相互作用。我们 提议通过生成针对细胞子集的新工具来提高细胞消融的资源 由双重表型标记定义的种群。使用条件目标的功能丧失研究 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 在体内，尽管它们仅针对一个标记标记的细胞群。高级方法正在改变， 加快我们识别和理解免疫，神经和其他细胞亚群的方式。这些 细胞亚群可以用多种表型标记标记。我们也预计会增长 在大脑倡议时代将发现免疫或神经亚群的光谱 后人类基因组项目时代。尽管当前的工具可用于消灭细胞人群，但它们 不能专门针对细胞亚群，因为它们只能靶向一个制造商，而不是双标记的细胞。 因此，有必要开发能够消除动物中细胞亚集的仪器以损失 - 功能研究。为此，我们建议生成一个Intermedilysin（ILY）介导的细胞亚群 消融模型。 Ily是由Intermedius链球菌（SI）分泌的一种毒素，仅结合人类细胞 膜蛋白CD59（HCD59），但对任何其他物种的CD59均不存在。一旦束缚，迅速而有力 裂解细胞。我们最近建立了CRE诱导的HCD59转基因小鼠系（IHCD59），其中HCD59 表达仅在CRE介导的重组后发生。给予iLy对各种CRE +/- IHCD59 +/-小鼠导致免疫，上皮或神经细胞的快速，特异性消融，而没有靶向 效果。我们还测试了几种疾病模型中的ILY/IHCD59介导的细胞消融方法 免疫细胞功能，肝细胞和/或胆道上皮损伤和再生以及神经细胞损伤。 这条线（IHCD59）仅针对一个标记标记的细胞种群，不能用于消融子集细胞 人口。为了进一步促进该工具朝着子集细胞消融迈进，我们建议开发和表征该工具 CAG（CMV早期增强剂/鸡β肌动蛋白）-floxp-stop-floxp-frt-stop-frt（dihcd59）或double 诱导小鼠应变，其中转基因（HCD59）表达仅在CRE-和FLP介导的 组合事件。具体来说，我们将研究我们的工作假设，即1）HCD59的表达将为 特异性在靶向亚群中由CRE和FLP介导的组合事件中的靶向亚群介导 DIHCD59或通过产生三重（CRE+/FLP+/DIHCD59+）转基因阳性小鼠； 2）注射 三元转基因阳性小鼠将消除细胞的亚群，以进一步研究其在体内的功能。