Humanizing Regulators of the Complement Cascade to Improve Research Relevance

人性化补体级联调节器以提高研究相关性

• 批准号: 9034796
• 负责人: Gareth R Howell
• 金额: $ 8.75万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9034796
• 关键词: Aging; Alleles; Alzheimer' s Disease; Alzheimer' s disease risk; Amino Acid Sequence; Animal Model; Antibodies; Astrocytes; Autoimmune Diseases; Autoimmunity; B-Lymphocytes; Binding; Biological Assay; Biological Process; Blood; Blood Cell Count; Blood Cells; Brain; Cells; Chromosomes; Collaborations; Communicable Diseases; Communities; Complement; Complement 1q; Complement 3d Receptors; Complement Activation; Complement Receptor; Complex; Cytolysis; Data; Dendritic Cells; Development; Disease; ES Cell Line; Endothelial Cells; Engineering; Ensure; Erythrocytes; Flow Cytometry; Follicular Dendritic Cells; Foundations; Functional disorder; Genes; Genetic; Genetic Engineering; Genetic Recombination; Genetic Transcription; Genetic Variation; Glaucoma; Health; Human; Human Engineering; Immune; Immune System Diseases; Immune response; Immunofluorescence Immunologic; In Situ Hybridization; Infection; Intergenic Sequence; Langerhans cell; Lectin; Letters; Lupus; Malaria; Membrane; Microglia; Modeling; Mouse Protein; Mouse Strains; Mus; Neurodegenerative Disorders; Neurons; Pathway interactions; Pattern; Phagocytosis; Plasma; Protein Isoforms; Proteins; RNA; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Series; Site; Skin; Southern Blotting; T-Lymphocyte; Technology; Testing; The Jackson Laboratory; Time; Transcript; Validation; Variant; Western Blotting; Work; age related; base; blastocyst; cell type; complement C3 precursor; embryonic stem cell; eosinophil; extracellular; genetic regulatory protein; genome wide association study; human disease; improved; macrophage; monocyte; mouse genome; mouse model; neuroinflammation; neutrophil; offspring; podocyte; promoter; public health relevance; response; species difference; tool; transmission process

 DESCRIPTION (provided by applicant): Activation of the complement cascade occurs in development, aging and a wide variety of human disease including neurodegenerative diseases, autoimmune diseases and in response to infections. Much of our understanding of the complement cascade has come from animal models. However, there are critical species differences between these models and humans that make data generated inadequate. This important difference hinders our ability to understand the complex role of the complement cascade and its key regulators in health and disease. In particular, there is no true functional equivalent of complement receptor 1 (CR1) that modulates the activity of complement components C1q, C3 and C4, key molecules in the complement cascade. Regulators of the complement cascade are also good targets for developing new therapies for diseases that involve complement activation. The closest functional equivalent to the human CR1 protein in mice is an isoform produced by the Cr2 gene, but its expression pattern differs substantially. Previous modeling of human CR1 in mice has been restricted to a limited number of cell types. Therefore, to overcome this major limitation of mice in modeling complement regulation, we have targeted mouse embryonic stem (ES) cells to express the human CR1 gene, driven by the human CR1 promoter and upstream sequences, and the human CR2 gene in place of the mouse Cr2 gene. We have further engineered these ES cells to enable site-specific recombination to generate two of the most common alleles of the human CR1 gene, the longer and the shorter forms. Correct targeting was confirmed, chimeric mice generated and germline transmission confirmed. Expression of CR1 and CR2 in both the blood and the brain was shown by RTPCR. In this proposal, we will fully determine the potential of this new strain to understand activation and regulation of the complement cascade in health and disease. We have two aims. In Aim 1, we will perform site-specific recombination to show we can generate an allelic series of CR1 and CR2 transcripts, including the common isoforms of human CR1. In Aim 2, we will use flow cytometry, immunofluorescence, RNA in situ hybridization and RT-PCR to determine the expression patterns of human CR1 and CR2 proteins. We will assess expression of CR1 and CR2 in specific cells in the blood, as this will be crucial in understanding the role of CR1 and CR2 in neuroinflammatory and immune disorders. Further, given the recent association of a region encompassing the CR1 gene in Alzheimer's disease (AD), we will determine the expression of CR1 transcripts and proteins in the brain. This validated mouse strain will lay the foundation for our work studying the role of complement in neurodegenerative disorders, particularly AD. It will also be made available without restrictions as we anticipate it being used by many to investigate the broader role of complement regulators in health and disease.

 描述（由申请人提供）：补体级联的激活发生在发育、衰老和多种人类疾病中，包括神经退行性疾病、自身免疫性疾病以及对感染的反应。我们对补体级联的大部分理解来自动物模型。然而，这些模型和人类之间存在关键的物种差异，导致生成的数据不足，这一重要差异阻碍了我们理解补体级联及其关键调节因子在健康和疾病中的复杂作用的能力。补体受体 1 (CR1) 的真正功能等同物，可调节补体成分 C1q、C3 和 C4（补体级联中的关键分子）的活性，也是开发补体激活相关疾病新疗法的良好靶点。小鼠体内与人类 CR1 蛋白功能最接近的同工型是 Cr2 基因产生的同工型，但其表达模式有很大不同，之前对小鼠体内人类 CR1 的建模仅限于有限数量的细胞类型。为了克服小鼠在补体调节建模中的这一主要限制，我们以小鼠胚胎干 (ES) 细胞为目标，表达由人 CR1 启动子和上游序列驱动的人 CR1 基因，并用人 CR2 基因代替小鼠 Cr2我们进一步设计了这些 ES 细胞，使其能够进行位点特异性重组，以生成人类 CR1 基因的两个最常见的等位基因，即更长和更短的形式，并确认了正确的靶向，生成了嵌合小鼠并确认了种系传播。 RTPCR 显示了血液和大脑中 CR1 和 CR2 的表达。在本提案中，我们将充分确定这种新菌株在了解健康和疾病中补体级联的激活和调节方面的潜力。在目标 1 中，我们将进行位点特异性重组，以证明我们可以生成一系列等位基因 CR1 和 CR2 转录本，包括人类 CR1 的常见亚型。在目标 2 中，我们将使用流式细胞术，我们将通过免疫荧光、RNA 原位杂交和 RT-PCR 来确定人类 CR1 和 CR2 蛋白的表达模式，因为这对于了解 CR1 和 CR2 的作用至关重要。此外，鉴于最近发现 CR1 基因区域与阿尔茨海默病 (AD) 相关，我们将确定 CR1 转录本和蛋白质在大脑中的表达，这种经过验证的小鼠品系将为我们的研究奠定基础。工作研究补体在神经退行性疾病，特别是 AD 中的作用。正如我们预期的那样，它也将不受限制地提供。 许多人研究补体调节剂在健康和疾病中的更广泛作用。