GENETICS OF HUMAN LYMPHOCYTE ANTIGEN CD8

描述(改编自《调查者摘要》)：细胞表面 糖蛋白CD8可作为T细胞分化为 细胞毒性/调节性细胞谱系。在CD8-α基因敲除小鼠中 谱系未能发展，表明CD8的关键功能作用。 这些T细胞上CD8的主要形式是CD8α/β 杂二聚体。相比之下，人类NK细胞和上皮内细胞的一部分 TCRγ/Delta的淋巴细胞只表达CD8α/α 同源二聚体。我们的目标是确定CD8-α和CD8-β之间的联系 基因在T细胞发育过程中作为模型系统受到基因调控 以了解T细胞分化。具体地说，我们计划找到 调节元件，如消音剂/增强剂和执行分子 分析分子标记基因，我们希望能够靶向 标记基因在表达CD8的细胞中的表达。关键监管 元素将通过DNase I超敏感图谱和DNase I超敏感图谱进行识别 使用转基因动物在体内测试基因组结构。我们计划 或者：(I)将带有超敏位点(HS)的区域连接到标记基因； 或(Ii)从人95kb片段中删除包含HS位点的区域，该片段 我们发现允许在转基因小鼠中表达CD8-β基因 CD8+T细胞，而不是CD4+T细胞。对于删除区域，我们将使用 一种非常适合处理大块的新型酵母/细菌载体 关于DNA的。胸腺细胞、成熟淋巴样细胞和 上皮内淋巴细胞将用单抗和流式细胞仪进行监测。 识别出的关键区域将被用来识别蛋白质 然后对结合蛋白进行鉴定。化验 建议包括DNase I足迹、EMSA和突变/功能 用报告基因进行分析。这项工作将使我们更好地理解 正常的T细胞分化构成了设计的基础 处理白血病和其他淋巴样肿瘤的策略 分化异常。我们应该能够识别出监管因素 这将允许将基因靶向细胞毒/抑制细胞。 这些细胞在体外有希望成为免疫治疗剂。 对患者进行致敏和注射。能够将基因定位于 这种血统可以增强发育这些细胞的能力 免疫疗法。

DESCRIPTION (Adapted from the Investigator's abstract): The cell surface glycoprotein CD8 serves as a marker for the differentiation of T-cells into the cytotoxic/regulatory cell lineage. In CD8-alpha knockout mice this lineage fails to develop indicating the critical functional role of CD8. The predominant form of CD8 on these T-cells is the CD8 alpha/beta heterodimer. In contrast, a subset of human NK cells and intraepithelial lymphocytes that are TCR gamma/delta exclusively express CD8 alpha/alpha homodimers. Our goal is to determine how the linked CD8-alpha and CD8-beta genes are genetically regulated during T-cell development as a model system for understanding T-cell differentiation. Specifically, we plan to locate regulatory elements such as silencer/enhancers and perform a molecular analysis of the elements to marker gene, we hope to be able to target expression of a marker gene to CD8 expressing cells. Critical regulatory elements will be identified by DNase I hypersensitivity mapping and by testing genomic constructs in vivo using transgenic animals. We plan to either: (i) link regions with hypersensitive sites (HS) to a marker gene; or (ii) delete regions containing HS sites from a human 95 kb fragment that we found allows for expression of the CD8-beta gene in transgenic murine CD8+ T-cells but not CD4+ T-cells. For deleting regions, we will make use of a new yeast/bacterial vector ideally suited for manipulating large pieces of DNA. Expression patterns on thymocytes, mature lymphoid cells, and intraepithelial lymphocytes will be monitored with mAbs and flow cytometry. The identified critical regions will be characterized to identify protein binding sites and to then characterize the binding proteins. Assays proposed include DNase I footprinting, EMSAs, and mutational/functional assays with reporter genes. This work will lead to a better understanding of normal T-cell differentiation which forms a basis for designing strategies to manipulate leukemias and other lymphoid tumors that have abnormal differentiation. We should be able to identify regulatory elements that will allow for the targeting of genes to cytotoxic/suppressor cells. These cells have promise as immunotherapeutic agents by in vitro sensitization and injection into patients. Being able to target genes to this lineage could enhance the ability to develop these cells for immunotherapy.