CHARACTERIZATION OF MURINE ACIDIC FGF GENE

鼠酸性 FGF 基因的表征

• 批准号: 3085427
• 负责人: Kevin Victor Hackshaw
• 金额: $ 7.7万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3085427
• 关键词: RNase protection assay; age difference; cell membrane; disease /disorder model; fibroblast growth factor; gene expression; genes; genetic mapping; genetic promoter element; genetic regulation; genetic transcription; inflammation; interleukin 1; laboratory mouse; leukotrienes; macrophage; northern blottings; nucleic acid hybridization; nucleic acid sequence; platelet derived growth factor; prostaglandins; protein structure function; site directed mutagenesis; systemic lupus erythematosus; tissue /cell culture

The objectives of this Physician Scientist Grant Application are for the candidate to (1) obtain an in depth basic science learning experience emphasizing a new discipline (Molecular Biology) while (2) conducting research under the direct supervision of a sponsor with extensive experience in this discipline. Subsequently, the candidate will apply these research tools to his current research interest in Phase II under the continuing supervision of his sponsor in conjunction with the advisory committee. The Phase I project will focus on characterization of murine acidic fibroblast growth factor gene. The specific aims are: (1) to determine the gene structure of mouse aFGF. The research plan involves isolation and characterization through sequencing of mouse genomic DNA clones for aFGF. Identification of the transcription initiation site(s) will be done by primer extension and ribonuclease protection assay. Analysis of potential differences in gene expression in young and old mice will proceed by examining aFGF production in young and old cells by Northern blotting and hybridization. Determination of regulatory mechanisms of the gene encoding aFGF, will involve identifying the functional promoter region by CAT assay, deletional mapping and site directed mutagenesis to identify critical nucleotides in the promoter/enhancer region. Characterization of the mouse gene is necessary in order to: [1] better study biologic questions pertaining to inflammation and tumorigenesis; and [2] initiate studies of site-directed gene targeting in mouse systems. During phase II, studies of this and other growth factors will be applied to a murine model of autoimmune disease, the MRL lpr/lpr mouse. We hypothesize that in lupus a disturbance in the production of cytokines (HBGFs), IL-1, and PDGF) and/or in the responsiveness of macrophages to cytokines results in anomalous immunological function. It is suggested further that by systematically analyzing macrophages for production of these cytokines, we hope to better understand the role they may have in the expression of autoimmunity. Using murine models of lupus, these studies seek to screen for the production of aFGF, bFGF, IL-1 and PDGF in macrophages from autoimmune and normal mice; to biochemically characterize the proposed cytokines in interest; to assess the biological characteristics of the protein(s) of interest i.e.; mitogenic activity, chemotactic activity; to examine the effects of culture with these cytokines on plasma membrane reorganization, prostanoid and leukotriene production; and to examine the regulatory mechanisms controlling the production of these factors. Examining these factors will allow us to better understand the role they have in promoting inflammation in this setting.

本医师科学家补助金申请的目的是为 候选人（1）获得深入的基础科学学习经验 强调一个新的学科（分子生物学），同时（2）进行 在申办者的直接监督下进行研究， 在这个学科的经验。 随后，考生将提出申请 这些研究工具，以他目前的研究兴趣，在第二阶段下， 与咨询一起继续监督他的赞助商 以马克思 第一阶段项目将集中在鼠酸性的表征， 成纤维细胞生长因子基因。 具体目标是：（1）确定 小鼠aFGF的基因结构。 研究计划包括隔离和 通过对aFGF的小鼠基因组DNA克隆进行测序进行表征。 转录起始位点的鉴定将通过 引物延伸和核糖核酸酶保护测定。 分析潜在 年轻小鼠和老年小鼠基因表达的差异将通过 通过北方印迹检测年轻和年老细胞中aFGF的产生， 杂交方法 确定基因编码的调控机制 aFGF，将涉及通过CAT测定鉴定功能性启动子区， 缺失定位和定点突变，以确定关键的 启动子/增强子区域中的核苷酸。 小鼠表征 基因是必要的，以便：[1]更好地研究生物学问题 与炎症和肿瘤发生有关;[2]开始研究 小鼠系统中的定点基因靶向。 在第二阶段， 这种和其它生长因子将应用于 自身免疫性疾病，MRL lpr/lpr小鼠。 我们假设在狼疮a中 细胞因子（HBGF）、IL-1和PDGF的产生紊乱）和/或 在巨噬细胞对细胞因子的反应性中， 免疫功能 进一步建议，通过系统地 分析巨噬细胞产生这些细胞因子，我们希望能更好地 了解它们在自身免疫表达中的作用。 使用 在狼疮的小鼠模型中，这些研究试图筛选 自身免疫和正常小鼠巨噬细胞中aFGF、bFGF、IL-1和PDGF; 生物化学表征所提出的感兴趣的细胞因子;评估 目标蛋白的生物学特性，即; 促有丝分裂活性、趋化活性;检查培养的影响 与这些细胞因子对质膜重组，前列腺素和 白三烯的产生;并检查调节机制 控制这些因素的生产。 研究这些因素将 让我们更好地了解它们在促进炎症中的作用， 在这种情况下。