Molecular Biology and Genetics Core

摘要： 这个分子生物学和遗传学核心(核心B)将提供基本的、尖端的、分子生物学和 遗传学专业知识，以促进这一高度集成的PPG专注于呼吸机的翻译影响- 诱发肺损伤(VILI)/急性呼吸窘迫综合征(ARDS)。PPG Core B将负责 启动子荧光素酶报告基因的生成及其修饰的启动子荧光素酶报告基因的构建 构造(连续删除或点突变)。核心B还将为KEY生成表达DNA质粒 在所有三个项目中对不同过度表达系统的蛋白质进行研究，包括E.Coli。系统(用于 核心D产生重组蛋白)，哺乳动物细胞系统(用于研究蛋白质功能的项目 内皮细胞或小鼠肺递送)。我们还将对这些基因进行定点突变(SDM) 表达载体引入代表优先单核苷酸多态(SNPs)的突变 或感兴趣的翻译后修饰(PTM)。核心B还将提供DNA的表观遗传学分析 用常规的分子生物学方法进行甲基化和乙酰化。作为验证步骤，核心B将执行 用凝胶迁移率改变分析(EMSA)和染色质研究DNA-蛋白质相互作用 免疫沉淀(CHIP)。此外，核心B将为项目调查人员提供一个特点良好的 ARDS生物库(即来自欧洲血统和非洲血统受试者的DNA)，选定的完整列表 每个PPG候选基因的SNPs、中通量基因分型服务和数据分析工具 PPG基因SNPs与VILI/ARDS易感性及严重程度的关系因此，总体来说，核心B 将利用这些技术为所有三个项目提供分子生物学和遗传学研究平台 并产生关于这一PPG研究目标基因的新信息。所有三个项目和 核心D将利用这个集中的核心进行PPG中计划的分子生物学和遗传学分析。

ABSTRACT: This Molecular Biology & Genetics Core (Core B) will provide essential, cutting edge, molecular biology and genetics expertise to facilitate the translational impact of this highly integrated PPG focused on ventilator- induced lung injury (VILI) / acute respiratory distress syndrome (ARDS). PPG Core B will be responsible for the generation of promoter luciferase reporter constructs, as well as modified promoter luciferase reporter constructs (serially deleted or point mutated). Core B will also generate expression DNA plasmids for key proteins studies in all three Projects for different over-expression systems including the E. Coli. system (for Core D to generate recombinant protein), mammalian cell system (for projects to study protein function in endothelial cells or murine lung delivery). We will also perform site-directed mutagenesis (SDM) on these expression constructs to introduce mutations representing prioritized single nucleotide polymorphisms (SNPs) or post-translational modifications (PTMs) of interest. Core B will also provide epigenetic analysis on DNA methylation and acetylation with routine molecular biology methods. As a validation step, Core B will perform DNA-protein interaction characterizations utilizing electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). In addition, Core B will provide Project investigators with a well-characterized ARDS biobank (i.e. DNA from European descent and African descent subjects), a complete list of selected SNPs for each PPG candidate gene, mid-throughput genotyping services, and data analysis tools to test the association between PPG selected SNPs and susceptibility and severity of VILI/ARDS. Thus, overall Core B will utilize these technologies to provide a molecular biology and genetics study platform for all three Projects and generate novel information on the genes targeted for investigation in this PPG. All three Projects and Core D will utilize this centralized core for the molecular biology and genetics analyses planned in this PPG.