Humanizing Regulators of the Complement Cascade to Improve Research Relevance

 描述(申请人提供)：补体级联的激活发生在发育、衰老和各种各样的人类疾病中，包括神经退行性疾病、自身免疫疾病和对感染的反应。我们对补体级联的大部分理解都来自动物模型。然而，这些模型与人类之间存在着关键的物种差异，这使得生成的数据不够充分。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。特别是，补体受体1(CR1)没有真正的功能等价物来调节补体成分C1q、C3和C4的活性，补体成分C1q、C3和C4是补体级联中的关键分子。补体级联的调节器也是开发涉及补体激活的疾病的新疗法的良好靶点。在小鼠中，与人类CR1蛋白功能最接近的等价物是Cr2基因产生的一种异构体，但其表达模式有很大不同。以前在小鼠身上建立的人类CR1模型仅限于有限数量的细胞类型。因此，为了克服小鼠在模拟补体调节方面的这一主要局限性，我们以小鼠胚胎干细胞为靶点，在人CR1启动子和上游序列的驱动下表达人CR1基因，并用人CR2基因取代小鼠的Cr2基因。我们进一步改造了这些ES细胞，使其能够进行定点重组，以产生人类CR1基因最常见的两个等位基因，即较长和较短的形式。确认了正确的靶向，产生了嵌合小鼠，并确认了生殖系传播。逆转录聚合酶链式反应显示CR1和CR2在血和脑中均有表达。在这项提案中，我们将充分确定这种新菌株的潜力，以了解补体级联在健康和疾病中的激活和调节。我们有两个目标。在目标1中，我们将进行位点特异性重组，以证明我们可以产生CR1和CR2转录本的等位基因系列，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、免疫荧光、RNA原位杂交和RT-PCR来确定人CR1和CR2蛋白的表达模式。我们将评估CR1和CR2在血液中特定细胞中的表达，因为这对于了解CR1和CR2在神经炎性和免疫疾病中的作用至关重要。此外，考虑到最近包含CR1基因的区域与阿尔茨海默病(AD)的关联，我们将确定CR1转录本和蛋白质在大脑中的表达。这个经过验证的小鼠品系将为我们研究补体在神经退行性疾病，特别是阿尔茨海默病中的作用奠定基础。它也将不受限制地提供，因为我们预计它将被使用 许多人调查补体调节因子在健康和疾病中的更广泛作用。

 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.