Bioassay Core

生物测定核心

• 批准号: 8795749
• 负责人: Pedro A. Jose
• 金额: $ 22.31万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8795749
• 关键词: ADP-ribosyl Cyclase; American; Binding Proteins; Biological Assay; Blood Pressure; Blood Vessels; Breeding; Capillary Electrophoresis; Cell Culture Techniques; Cessation of life; Control Animal; DNA; Dopamine; Ensure; Enzymes; Essential Hypertension; Fibroblast Growth Factor; Gene Deletion; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genotype; Goals; Hypertension; Instruction; Kidney; Knock-out; Measures; Membrane; Messenger RNA; Methods; Mus; NADPH Oxidase; Nitrates; Nitrites; Oxidation-Reduction; Oxidative Stress; Paraoxonase-2; Program Research Project Grants; Proteins; Quality Control; RNA; Reactive Oxygen Species; Research; Resources; Risk Factors; Role; Services; Small Interfering RNA; Smooth Muscle Myocytes; Sulfhydryl Compounds; Superoxide Dismutase; Superoxides; Techniques; Technology; Transgenic Animals; Transgenic Mice; Transgenic Organisms; animal breeding; catalase; extracellular; global health; in vivo; interest; knockout gene; mRNA Expression; programs; protein expression; receptor; small hairpin RNA

The Bioassay Core (Core C) will provide services to all three projects in this program project grant (PPG) and will be directed by Dr. Kathryn Sandberg. The overall goal of this core is to provide analytical support for each project of the PPG by aiding in gene silencing and gene expression through si_RNA and lentiviral strategies and to ensure quality control of employed strategies by genotyping knockout and transgenic (tg) animals and by measuring mRNA and protein expression of targeted genes of interest. Genotyping will be performed on single-gene deleted mice and at least two strains of transgenic mice bred in Core B including: gene deletions of extracellular superoxide dismutase (SOD-3); NADPH oxidase-4 (NOX-4); p47'"^¿'', a critical subunit of NADPH oxidase-2 (p47'"'¿''); ADP ribosyl cyclase 38 (CD38); fibroblast growth factor binding protein-1 (FGF-BP1) and -3 (FGF-BP3); dopamine-2 receptor (D2R); paraoxonase-2 (PON-2); overexpr-ession in vascular smooth muscle cells of p22''^¿'*, a membrane subunit of NOX (VSMCP^^"'^"'' tg) and catalase (VSMC^^' tg). This core will also determine levels of oxidative stress by measuring nitrates and nitrites, superoxide and the redox status of thiols by enzyme assays and capillary electrophoresis. Furthermore, this core is poised to develop new methods for gene manipulation for each project as gene manipulation technologies advance in the field. Aim 1 will construct and develop gene silencing techniques including siRNA and lentiviral approaches to study the role of genes of interest both in vivo and in cell culture. Aim 2 will assess gene expression at the DNA, mRNA and/or protein levels to ensure quality control of animal breeding performed in Core B and the efficacy of gene manipulation strategies in each of the three projects including via gene knockout, siRNA and shRNA-lentiviral approaches. Aim 3 will determine the degree of oxidative stress by measuring nitrates, nitrites, reactive oxygen species (ROS) and the redox status of thiols in each of the three projects. RELEVANCE (See instructions): Hypertension is a global health concern. Fifty million Americans have hypertension that requires treatment and over 1 billion people woridwide have hypertension. In fact, suboptimal blood pressure is the number one risk factor for death throughout the worid. Though much research has been conducted on hypertension, the mechanisms underiying the main cause of hypertension, i.e., essential hypertension, remain unknown. This Core serves a crucial resource for this program project that is devoted to understanding the role of oxidative stress in hypertension.

生物测定核心（核心C）将为本计划项目赠款（PPG）中的所有三个项目提供服务 并将由凯瑟琳·桑德伯格（Kathryn Sandberg）博士执导。该核心的总体目标是为 PPG的每个项目通过Si_RNA和慢病毒来帮助基因沉默和基因表达 策略并通过基因分型敲除和转基因（TG）来确保就业策略的质量控制 动物以及测量靶向基因的mRNA和蛋白质表达。基因分型将是 在单基因删除的小鼠和在Core B中繁殖的至少两种转基因小鼠进行了进行：包括： 细胞外超氧化物歧化酶（SOD-3）的基因缺失； NADPH氧化酶4（NOX-4）; p47'“^€''，一个批判 NADPH氧化酶-2的亚基（p47'“'€'）； ADP核糖基环酶38（CD38）；成纤维细胞生长因子结合 蛋白-1（FGF-BP1）和-3（FGF-BP3）;多巴胺-2受体（D2R）;副氧合酶2（PON-2）; p22''''*的血管平滑肌细胞中的过度分发，nox的膜亚基（vsmcp ^^“''''''''''''''''''' 过氧化氢酶（vsmc ^^'tg）。该核心还将通过测量硝酸盐和 酶测定和毛细管电泳的亚硝酸盐，超氧化物和硫醇的氧化还原状态。 此外，该核心被中毒以开发每个项目的基因操纵的新方法，作为基因 操纵技术在现场发展。 AIM 1将构建和开发基因沉默技术 包括siRNA和慢病毒方法研究感兴趣基因在体内和细胞中的作用 文化。 AIM 2将评估DNA，mRNA和/或蛋白质水平的基因表达，以确保质量控制 在核心B中进行的动物育种以及三种基因操纵策略的效率 包括通过基因敲除，siRNA和shRNA慢病毒方法在内的项目。 AIM 3将确定 通过测量硝酸盐，硝酸盐，活性氧（ROS）和氧化还原，氧化应激程度 三个项目中的每个项目中的每个硫醇的状态。 相关性（请参阅说明）： 高血压是全球健康问题。五百万美国人患有高血压，需要治疗 超过10亿人的Wonderwide患有高血压。实际上，次优血压是第一 在整个战争中死亡的危险因素。尽管已经对高血压进行了许多研究 高血压主要原因（即本质高血压）的主要原因的机制仍然未知。这 核心为该计划项目提供了至关重要的资源，该项目致力于了解氧化的作用 高血压的压力。