A novel and improved mouse system for studying macrophage specific gene deletion

一种用于研究巨噬细胞特异性基因缺失的新型改良小鼠系统

• 批准号: 8038759
• 负责人: Harris R Perlman
• 金额: $ 22.88万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-15 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038759
• 关键词: Ablation; Adult; Affect; Alleles; Animals; Automobile Driving; Biological; Biological Assay; Biology; Bone Marrow; Breeding; Cells; Communicable Diseases; Communities; Development; Disadvantaged; Disease; Engineering; Enhancers; Enterobacteria phage P1 Cre recombinase; Event; Flow Cytometry; Fluorescence; Gene Deletion; Genes; Genetic Recombination; Goals; Histocompatibility Testing; Homeostasis; Human; ITGAM gene; Immune; Immune response; Immunity; Infection; Inflammation; Inflammatory; Inflammatory Response; Internal Ribosome Entry Site; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Malignant Neoplasms; Masks; Mediating; Messenger RNA; Molecular; Molecular Biology; Muramidase; Mus; Myelogenous; Pathway interactions; Peritonitis; Phagocytes; Phenotype; Play; Population; Process; Production; Proteins; Reporter; Reporter Genes; Research; Research Personnel; Role; Site; Sorting - Cell Movement; Specificity; System; Thioglycolates; Time; Tissues; Tomatoes; Transcript; Transgenic Mice; Transgenic Organisms; Validation; Work; base; cell type; cytokine; gene function; granulocyte; improved; in vivo; macrophage; magnetic beads; novel; peripheral blood; prevent; promoter; recombinase; response; tool

DESCRIPTION (provided by applicant): Macrophages are crucial for tissue homeostasis and play essential roles in inflammation, immunity and cancer, and therefore understanding macrophage biology is fundamental for understanding homeostasis and disease. Currently available mice that target Cre expression for the deletion of floxed genes to macrophages have significant and severe restrictions, primarily based on the promoter driving Cre expression. 1) all of the available promoters currently driving Cre in macrophages are active in the entire myeloid lineage and even other cell types, which is frequently stronger than the one in macrophages, and therefore lack macrophage specificity; 2) currently available promoters only target a subset of macrophages; and 3) the two most commonly used Cre lines driven by F4/80 and LysM, are also knocked in one endogenous allele resulting in weak expression and prevent breeding of homozygous mice. The goal of this study is to develop and characterize a novel and improved transgenic mouse line to specifically target expression of Cre to macrophages. We propose to use the human CD68 promoter in combination with a macrophage-specific enhancer to drive expression of an activity-improved Cre and the dtTomato fluorescent protein from a bicistronic transcript for in vivo tracking and sorting. Transgenic mice will be extensively characterized with reporter genes and endogenous floxed genes and compared to the currently available Cre-expressing mice. Therefore our proposal to develop a novel true macrophage selective and specific Cre expressing mouse line in the C57BL/6 strain, and to make this mouse widely available to the research community, will significantly impact our current ability to study macrophages and will have significant ramifications for researchers studying macrophages in homeostasis, immunity and cancer, and will therefore enable the field to progress forward. PUBLIC HEALTH RELEVANCE: Macrophages are essential for tissue homeostasis and contribute to disease. Therefore understanding macrophage biology is fundamental to advance current knowledge of these processes, which requires macrophage specific deletion of genes using the Cre recombinase. Because currently available mice expressing Cre in macrophages have severe disadvantages, our study will develop and characterize a new and substantially improved transgenic mouse with macrophage specific expression of an improved version of Cre, which will be shared with the scientific community.

描述（由申请人提供）：巨噬细胞对于组织稳态至关重要，并在炎症、免疫和癌症中发挥重要作用，因此了解巨噬细胞生物学是了解稳态和疾病的基础。目前可用的针对 Cre 表达以删除巨噬细胞 floxed 基因的小鼠具有显着且严格的限制，主要基于驱动 Cre 表达的启动子。 1）目前在巨噬细胞中驱动Cre的所有可用启动子在整个骨髓谱系甚至其他细胞类型中都有活性，其通常比巨噬细胞中的启动子更强，因此缺乏巨噬细胞特异性； 2) 目前可用的启动子仅针对巨噬细胞的一个子集； 3) 由 F4/80 和 LysM 驱动的两种最常用的 Cre 系也被敲除一个内源等位基因，导致表达较弱并阻止纯合小鼠的繁殖。本研究的目的是开发和表征一种新型且改良的转基因小鼠品系，以特异性靶向巨噬细胞表达 Cre。我们建议将人 CD68 启动子与巨噬细胞特异性增强子结合使用，以驱动双顺反子转录物中活性改善的 Cre 和 dtTomato 荧光蛋白的表达，用于体内跟踪和分选。转基因小鼠将通过报告基因和内源性 floxed 基因进行广泛的表征，并与目前可用的 Cre 表达小鼠进行比较。因此，我们建议在 C57BL/6 品系中开发一种新型真正的巨噬细胞选择性和特异性 Cre 表达小鼠系，并使该小鼠广泛应用于研究界，这将显着影响我们目前研究巨噬细胞的能力，并将对以下方面产生重大影响：研究人员研究巨噬细胞在体内平衡、免疫和癌症中的作用，因此将使该领域不断向前发展。 公共卫生相关性：巨噬细胞对于组织稳态至关重要，并导致疾病。因此，了解巨噬细胞生物学对于推进当前对这些过程的了解至关重要，这需要使用 Cre 重组酶对巨噬细胞进行特异性基因删除。由于目前可用的在巨噬细胞中表达 Cre 的小鼠具有严重的缺点，因此我们的研究将开发并表征一种新的且显着改进的转基因小鼠，其巨噬细胞特异性表达改进版本的 Cre，并将其与科学界共享。