Generation of Cre/lox Mice for Inducible Deletion of PKC-epsilon in the Immune System

产生用于诱导删除免疫系统中 PKC-ε 的 Cre/lox 小鼠

• 批准号: 10057079
• 负责人: Michelle R Lennartz
• 金额: $ 8.14万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-10 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10057079
• 关键词: Alzheimer' s Disease; Animals; Antigen-Antibody Complex; Apoptotic; Area; Atherosclerosis; Attention; Binding; Blood Vessels; Breeding; Cells; Data; Defect; Development; Disease; Disease Progression; Disease model; Endothelial Cells; Environment; Family; Fertilization in Vitro; Fibrosis; Flow Cytometry; Foam Cells; Future; Generations; Genes; Human; ITGAM gene; Image; Immune; Immune System Diseases; Immune system; Immunofluorescence Immunologic; Impairment; In Vitro; Infection; Inflammation; Inflammatory; Knock-out; Knockout Mice; Leukotriene B4; Link; Lipid Inclusion; Lipids; LoxP-flanked allele; Malignant Neoplasms; Modeling; Morphology; Mouse Strains; Mus; Myelogenous; Myeloid Cells; NF-kappa B; Necrosis; Neurologic; Oils; PRKCA gene; Pathway interactions; Phagocytes; Phagocytosis; Phenotype; Plant Roots; Play; Production; Property; Protein Isoforms; Reagent; Reporter; Research Personnel; Respiratory Burst; Role; Scleroderma; Serum; Serum Markers; Siblings; Signal Transduction; Smooth Muscle Myocytes; Stains; TAL1 gene; TNF gene; Thick; Tissues; Virus; Western Blotting; arm; cell type; chronic inflammatory disease; cytokine; feeding; gain of function; human disease; hypercholesterolemia; in vivo; inflammatory marker; interest; link protein; lipid mediator; macrophage; monocyte; mutant; neutrophil; novel; offspring; oxidized low density lipoprotein; prevent; protein kinase C epsilon; protein kinase D; recombinase-mediated cassette exchange; response; tool; virtual; wound healing

PROJECT SUMMARY In a family of 11 isoforms, protein kinase C-epsilon (PKC-) has unique binding and regulatory properties that cannot be compensated for by other PKCs. It is involved in neurological, vascular, and wound healing as evidenced from studies in the global PKC- knockout mouse. Notably, the global KO has underlying immune defects that prevent homozygous breeding and PKC- KO mice succumb to infections cleared by their wild type counterparts. As virtually every disease has an immune/inflammatory component, the results from in vivo studies with the KO mouse must be interpreted in the context of the unknown effects of the impaired immune system. Thus, there is an unmet need for a “cleaner” mouse in which to study the role of PKC- in disease, one in which PKC- can be deleted in a tissue specific manner. That is, a PKC-loxP/loxP (flox’d) mouse. This application will generate a PKC- floxed mouse, which, when crossed to a tissue-selective Cre, will specifically delete PKC- in the cells of choice. The significance is that PKC- flox’d mice will have an intact immune system that, when crossed to a Cre of choice, will produce offspring lacking PKC- only in the tissue of interest. The PKC- flox’d mouse can be considered a gateway strain, providing a tool for other investigators to study PKC- in their model of choice independently of the immune defects documented for the global knockout. We will cross them to reporter mice expressing lox-STOP-lox ZsGreen and LysM-Cre. The resulting mouse will have PKC- selectively deleted in macrophages (MØ) and neutrophils, which will also express the ZsGreen reporter. Our preliminary data indicate that MØ from PKC- mice accumulate more, and larger, lipid droplets and produce more TNF-, but less ResolvinD1, in response to lipid feeding or immune complexes. We hypothesize that PKC- expression in MØ will slow atherosclerosis development. In vivo, we will examine the role of MØ PKC- in the AAV8-PCSK9 model of atherosclerosis, using PKC-ef/fLysM-Cre+/± mice. AAV8- PCSK9 is a gain of function virus expressing a mutant that produces hypercholesterolemia and atherosclerosis in mice. The mutant PCSK9 gene is expressed in humans with hypercholesterolemia, bringing translational relevance to the model. Aortic root plaques from WT and PKC-f/fLysM-Cre+/± mice will be scored for metrics of plaque stability applied to the human disease. Imaging will be used to quantify the number and localization of plaque MØ, their polarization state (immunofluorescence for markers of M1, M2, and Mox) and lipid content (Oil Red O staining). Additionally, serum levels of cytokines and lipid mediators will be quantified. In vitro studies will identify specific steps in foam cell formation in which PKC- acts and will define the signature of cytokines and lipid mediators produced by WT and PKC-f/fLysM-Cre+/± MØ in response to immune complexes and apoptotic cells. In conclusion, we have identified PKC- as a novel player in regulating lipid retention and inflammation in an atherogenic environment. These studies will identify atherosclerosis-relevant pathways that are regulated by macrophage PKC-, pathways that can be examined in detail in a future R01.

项目摘要 在一个由11种亚型组成的家族中，蛋白激酶C-κ B（PKC-κ B）具有独特的结合和调节特性， 不能被其他PKC补偿。它参与神经，血管和伤口愈合， 这一点从全球PKC β基因敲除小鼠的研究中得到了证实。值得注意的是，全球KO具有潜在的免疫性， 阻止纯合繁殖的缺陷和PKC-PKKO小鼠死于由其野生型清除的感染， 类型对应物。由于几乎每种疾病都有免疫/炎症成分，因此体内结果 KO小鼠的研究必须在免疫功能受损的未知影响的背景下进行解释。 系统因此，对于研究PKC β 2在疾病中的作用的“清洁”小鼠存在未满足的需求， 其中PKC-β可以以组织特异性方式缺失。也就是说，PKC-IoxP/loxP（flox'd）小鼠。这 应用将产生PKC-Cre floxed小鼠，当与组织选择性Cre杂交时， 删除所选细胞中的PKC-β。重要的是，PKC抑制剂诱导的小鼠将具有完整的免疫系统， 系统，当杂交到一个Cre的选择，将产生后代缺乏PKC-β只在组织中， pkc-pkflox的小鼠可以被认为是一个网关菌株，为其他研究人员提供了一个工具。 在他们选择的模型中研究PKC-β，独立于全球记录的免疫缺陷。 漂亮我们将它们与表达lox-STOP-lox ZsGreen和LysM-Cre的报告基因小鼠杂交。所得 小鼠的巨噬细胞和中性粒细胞中的PKC-β选择性缺失，这些巨噬细胞和中性粒细胞也表达PKC-β。 ZsGreen记者。我们的初步数据表明，来自PKC抑制剂小鼠的MMP 2积累了更多，更大的脂质， 液滴和产生更多的TNF-α，但更少的ResolvinD 1，以响应脂质喂养或免疫复合物。我们 假设MØ中PKC-γ的表达将减缓动脉粥样硬化的发展。在体内，我们将检查 使用PKC-ef/fLysM-Cre+/±小鼠，MMPKC-CRs在动脉粥样硬化的AAV 8-PCSK 9模型中的作用。AAV8- PCSK 9是一种功能获得性病毒，表达一种可产生高胆固醇血症和动脉粥样硬化的突变体 对小鼠突变型PCSK 9基因在高胆固醇血症患者中表达， 与模型相关。将对来自WT和PKC-Crrf/fLysM-Cre+/±小鼠的主动脉根部斑块的以下度量进行评分： 应用于人类疾病的斑块稳定性。将使用成像来量化 斑块MMPs、其偏振状态（M1、M2和Mox标记物的免疫荧光）和脂质含量（油 红O染色）。此外，将定量细胞因子和脂质介质的血清水平。体外研究 将确定泡沫细胞形成的具体步骤，其中PKC-β起作用，并将确定细胞因子的特征 以及WT和PKC-β f/fLysM-Cre+/± Mc应答免疫复合物产生的脂质介质， 凋亡细胞总之，我们已经确定PKC-β是调节脂质潴留的一个新的参与者， 动脉粥样硬化环境中的炎症。这些研究将确定动脉粥样硬化相关的途径， 是由巨噬细胞PKC-β调节的，这些途径可以在未来的R 01中详细研究。