MV-ligase probes for visualization of apoptotic clearance

描述（由申请人提供）：我们合成了一种重组形式的拟病毒（MV）DNA拓扑异构酶1B蛋白，该蛋白具有不寻常的5 'OH连接活性（MV-连接酶）。这允许用于检测凋亡细胞清除的新型探针。在本计画中，我们将开发这种新的萤光探针，以进行原位研究。探针将仅显示吞噬和消化凋亡细胞DNA的任何谱系的那些活性吞噬细胞（专业、业余吞噬细胞或周围组织细胞）。在亚细胞水平，探针将 是激活的吞噬溶酶体的第一个特异性标志物-细胞器执行凋亡细胞尸体的实际吞噬降解。虽然吞噬阶段是有效执行凋亡的关键，目前还没有特异性的吞噬溶酶体消化凋亡细胞的原位探针。该项目将缩小这一技术差距，并将提供一种有用的分子工具。它将创造一种有利的产品，广泛适用于病理学的研究，其中死亡细胞的清除是必不可少的，如癌症，炎症，感染和自身免疫疾病。该提案的具体目的是：1）开发基于MV-连接酶的探针，其将选择性地标记吞噬凋亡核的细胞的吞噬溶酶体，并使用体外模型测试探针。2)开发第一个原位检测方法，用于靶向检测组织切片中的凋亡细胞清除，并测试和优化其在几种凋亡模型中的灵敏度、特异性和适用性。

DESCRIPTION (provided by applicant): We synthesized a recombinant version of Mimivirus (MV) DNA topoisomerase 1B protein possessing an unusual 5'OH ligation activity (MV-ligase). This permits a novel type of probe for the detection of apoptotic cell clearance. In this project w will develop such new fluorescent probes for in situ research. The probes will visualize only those active phagocytic cells of any lineage (professional, amateur phagocytes or surrounding tissue cells) which engulf and digest apoptotic cell DNA. At the subcellular level, the probes will be the first specific markers of activated phagolysosomes - cellular organelles performing the actual phagocytic degradation of apoptotic cell corpses. Although the phagocytic phase is critical for efficient execution of apoptosis, there are currently no in situ probes specific for phagolysosomes digesting apoptotic cells. The project will close this technological gap and will provide a useful molecular tool. It will create an advantageous product with wide applicability in studies of pathologies where clearance of dying cells is essential, such as cancers, inflammation, infection and auto-immune disorders. The Specific Aims of the proposal are: 1) to develop the MV-ligase-based probes that will selectively label phagolysosomes of the cells that engulfed apoptotic nuclei and to test the probes using in vitro models. 2) To develop the first in situ assay for targeted detection of apoptotic cell clearance in tissue sections and to test and optimize its sensitivity, specificity and applicability in several apoptotic models.