New Tools to Understand Microglial Function

描述（由申请人提供）：小胶质细胞是脑内髓系来源的常驻细胞，但它们在健康和疾病中的确切作用仍然知之甚少。无法可靠地区分小胶质细胞和密切相关的被称为巨噬细胞的髓样细胞，这混淆了许多关于小胶质细胞功能的研究。因此，我们筛选了一种新的小胶质细胞特异性基因，可以可靠地识别、靶向和表征小胶质细胞。在我们的初步研究中，我们发现TM119是一种高表达的跨膜蛋白，是一种高表达的小胶质细胞特异性标志物，不被巨噬细胞或其他外周免疫细胞表达。在这个应用中，我们将开发几种基于TM119表达的新工具来选择性地鉴定、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TM119的抗体，用于免疫染色鉴定小胶质细胞和免疫计划从全脑组织中分离纯小胶质细胞。在第二个目标中，我们将开发两个小鼠系，一个TM119/Cre重组酶敲入小鼠和一个TM119/CreERT2 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（敲入型）或诱导型（BAC） Cre表达。将这些小鼠与其他表达Cre依赖基因的小鼠系杂交，将允许对小胶质细胞基因进行选择性和特异性操作。在最终目标中，我们将使用目标1和目标2中开发的抗体和遗传工具，从成年、发育或炎症小鼠组织中急性纯化小胶质细胞，通过基因阵列和RNAseq生成纯小胶质细胞的基因图谱。这些档案和所有开发的工具将立即提供给出版感兴趣的研究人员。在拟议的研究中开发的工具将使研究人员能够更好地理解小胶质细胞的作用，这可能对更好地理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.