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MOLECULAR CHARACTERIZATION OF PERFORIN GENE REGULATION

穿孔素基因调控的分子特征

基本信息

批准号：
6375921
负责人：
Mathias Georg Lichtenheld
金额：
$ 21.96万
依托单位：
UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-09-01 至 2004-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6375921
关键词：
DNA footprinting T cell receptor cytotoxic T lymphocyte genetic regulation genetic regulatory element genetically modified animals interleukin 2 laboratory mouse leukocyte activation /transformation natural killer cells pore forming protein tissue /cell culture transfection

项目摘要

DESCRIPTION (Adapted from Investigator's abstract): Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) defeat immunogenic tumors and virus infections. Physiologically, most CTL reside in the body as pre-effectors (pre-CTL) that are unable to kill. When the immune system is confronted with intracellular pathogens, preCTL are expanded and armed. During this process many genes are newly expressed or upregulated in preCTL, leading to their proliferation and differentiation into effector CTL(eff CTL). One of these genes is perforin, a membrane "perforating" killer molecule essential for the function of the adaptive immune system. In contrast, NK cells, perhaps best thought of as primordial CTL that lack receptors for a specific antigen, constitutively express perforin and kill without activation. Our experiments have shown that perforin gene expression is determined at the transcriptional level and that this process requires regulatory domains in addition to the promoter. This application will continue to identify and characterize these regulatory domains by elucidating constitutive and inducible nuclear events at the perforin gene locus in NK cells and CTL. Molecules involved in this process ultimately may be of therapeutical use or may become therapeutical targets. From the immunological and developmental point of view, the regulatory domains ultimately may shed light on transcription factors that control not only the perforin gene but also the commitment to and progression of the perforin expressing lineages during hematolymphopoiesis. This application specifically proposes to: 1) explore the presence of additional regulatory domains within the boundaries of an active perforin gene locus in tumor cell lines; 2) characterize the function of potential regulatory domains in tumor cell lines; and 3) demonstrate the function of the regulatory domains in normal cells. To achieve these aims we will: 1) perform a DNase I sensitivity and hypersensitivity analysis of the perforin gene locus; 2) analyze potential regulatory domains by reporter gene assays; and 3) generate and analyze mice that are transgenic for regulatory DNAs identified in the first two aims.

描述(改编自《调查者摘要》)：自然杀手(NK) 细胞和细胞毒性T淋巴细胞(CTL)击败免疫原性肿瘤和病毒感染。从生理上讲，大多数CTL作为前效应物驻留在体内。 (CTL前)无法杀人的人。当免疫系统面对在细胞内病原体的作用下，前CTL被扩张并武装起来。在此期间过程许多基因在前CTL中新表达或上调，导致它们的增殖和分化为效应细胞集落刺激因子(EffCTL)。其中之一这些基因就是穿孔素，它是一种膜“穿孔”杀手分子适应性免疫系统的功能。相比之下，NK细胞，也许最好的想法是原始的CTL缺乏特定的受体抗原，结构性表达穿孔素，无需激活即可杀伤。我们的实验表明，穿孔素基因的表达是由转录水平，这一过程需要调控结构域在除了发起人。此应用程序将继续识别和通过阐明构成域和结构域来描述这些调控域 NK细胞和CTL穿孔素基因座可诱导的核事件。参与这一过程的分子最终可能具有治疗用途或可能成为治疗靶点。从免疫学和发育学角度从观点来看，监管领域最终可能会揭示转录因子不仅控制穿孔素基因，而且还控制穿孔素表达谱系的承诺和进展血淋巴生成。本申请特别提出：1)探索在边界内存在额外的监管领域肿瘤细胞系中活性穿孔素基因的定位；2)功能特征肿瘤细胞系中潜在的调控结构域；以及3)展示正常细胞中调节结构域的功能。为了实现这些目标我们将：1)执行DNase I敏感性和过敏性分析穿孔素基因座；2)利用报告分析潜在的调控结构域基因分析；以及3)产生和分析转基因小鼠前两个目标中确定的监管DNA。