New Tools to Understand Microglial Function

描述（由申请人提供）：小胶质细胞是脑内髓源性驻留细胞，但它们在健康和疾病中的确切作用仍知之甚少。无法可靠地区分小胶质细胞与称为巨噬细胞的密切相关的骨髓细胞混淆了许多小胶质细胞功能的研究。因此，我们筛选了一个新的小胶质细胞特异性基因，这将允许可靠的识别，靶向和表征小胶质细胞。在我们的初步研究中，我们确定TM 119，一种高表达的跨膜蛋白，作为一种高表达的小胶质细胞特异性标志物，不表达的巨噬细胞或其他外周免疫细胞。在本申请中，我们将开发几种基于TM 119表达的新工具来选择性地识别、分离和操纵小胶质细胞。在第一个目标中，我们将开发两种针对TM 119的抗体，用于通过免疫染色鉴定小胶质细胞，并通过免疫淘洗从全脑组织中分离纯的小胶质细胞。在第二个目标中，我们将建立两个小鼠品系，一个是TM 119/Cre重组酶基因敲入小鼠，一个是TM 119/CreERT 2 BAC转基因小鼠。这些小鼠将在小胶质细胞内选择性地驱动组成型（敲入）或诱导型（BAC）Cre表达。将这些小鼠与表达Cre依赖性基因的其他小鼠品系杂交将允许选择性和特异性操纵小胶质细胞基因。在最终目标中，我们将使用目标1和2中开发的抗体和遗传工具从成年、发育或发炎的小鼠组织中急性纯化小胶质细胞，以通过基因阵列和RNAseq生成纯小胶质细胞的基因谱。这些概况和开发的所有工具一经出版，将立即提供给感兴趣的研究人员。在拟议的研究中开发的工具将使研究人员能够更好地了解小胶质细胞的作用，这可能对更好地了解人类神经系统疾病的病理生理学和治疗至关重要。

DESCRIPTION (provided by applicant): Microglia is myeloid-derived resident cells within the brain but their exact roles in health and disease are still poorly understood. The inability to reliably distinguish microglia from closely related myeloid cells called macrophages confounds many studies of microglial function. Therefore we screened for a new microglial specific gene that would allow the reliable identification, targeting, and characterization of microglia. In our preliminary studies, we identify TM119, a highly expressed transmembrane protein, as a highly expressed microglia-specific marker that is not expressed by macrophages or other peripheral immune cells. In this application, we will develop several new tools based on TM119 expression to selectively identify, isolate, and manipulate microglia. In the first aim, we will develop two antibodies against TM119 for the identification of microglia by immunostaining and isolation of pure microglia from whole brain tissue by immunopanning. In the second aim, we will develop two mouse lines, a TM119/Cre recombinase knock-in mouse and a TM119/CreERT2 BAC transgenic mouse. These mice will drive constitutive (knock-in) or inducible (BAC) Cre expression selectively within microglia. Crossing these mice with other mouse lines that express Cre- dependent genes will allow selective and specific manipulation of microglial genes. In the final aim, we will use the antibody and genetic tools developed in Aims 1 & 2 to acutely purify microglia from adult, developing, or inflamed mouse tissues to generate gene profiles of pure microglia by gene array and RNAseq. These profiles and all tools developed will be made immediately available upon publication to interested researchers. The tools developed in the proposed studies will enable investigators to better understand the roles of microglia, which may prove critical for a better understanding of the pathophysiology and treatment of human neurological diseases.