Molecular Biology and Genetics Core

分子生物学和遗传学核心

• 批准号: 10094242
• 负责人: Joe G. N. Garcia
• 金额: $ 22.72万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-05 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094242
• 关键词: Acetylation; Acute Lung Injury; Adult Respiratory Distress Syndrome; African; Biological Assay; Biotechnology; Candidate Disease Gene; DNA; DNA Methylation; DNA-Protein Interaction; Data Analyses; Development; Electrophoretic Mobility Shift Assay; Endothelial Cells; Epigenetic Process; Escherichia coli; European; Gene Targeting; Generations; Genes; Genetic; Genetic Transcription; Genetic study; Genotype; Histone Acetylation; Infrastructure; Investigation; Laboratories; Luciferases; Lung; Mammalian Cell; Methods; Molecular Biology; Molecular Genetics; Mus; Mutate; Mutation; Patients; Post-Translational Protein Processing; Predisposition; Proteins; Receptor Signaling; Recombinant Proteins; Regulation; Reporter; Research Personnel; Role; SOX18 gene; Services; Severities; Single Nucleotide Polymorphism; Site-Directed Mutagenesis; Sphingosine-1-Phosphate Receptor; System; TLR4 gene; Technology; Testing; Validation; Ventilator-induced lung injury; biobank; chromatin immunoprecipitation; experimental group; genetic analysis; insight; interest; novel; overexpression; plasmid DNA; promoter; protein function; tool; translational impact

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.

抽象的： 这种分子生物学和遗传学核心（核心B）将提供必不可少的，最前沿，分子生物学和 遗传学专业知识，以促进这种高度综合PPG的翻译影响，该PPG专注于呼吸机 - 诱导肺损伤（VILI） /急性呼吸窘迫综合征（ARDS）。 PPG核心B将负责 启动子荧光素酶报告基因构建体以及改良的启动子荧光素酶报告基因的生成 构造（串行删除或点突变）。核B还将生成键的表达DNA质粒 蛋白质在所有三个项目中研究包括大肠杆菌在内的不同表达系统。系统（用于 核心D生成重组蛋白），哺乳动物细胞系统（用于研究蛋白质功能的项目 内皮细胞或鼠肺递送）。我们还将在这些方面执行定向的诱变（SDM） 表达构造以引入代表优先级单核苷酸多态性（SNP）的突变 或关注后翻译后修改（PTM）。核心B还将对DNA进行表观遗传分析 常规分子生物学方法的甲基化和乙酰化。作为验证步骤，核心B将执行 使用电泳迁移率分析（EMSA）和染色质的DNA蛋白相互作用表征 免疫沉淀（芯片）。此外，核心B将为项目调查人员提供良好的特征 ARDS BioBank（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，中期基因分型服务和数据分析工具的SNP PPG选定的SNP与VILI/ARDS的敏感性和严重程度之间的关联。因此，总体核心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 并生成有关在此PPG中进行研究的基因的新信息。这三个项目以及 核心D将利用这种集中核心进行分子生物学和遗传学分析，并在此PPG中计划。