Molecular Biology/Virus Production

分子生物学/病毒生产

• 批准号: 10657396
• 负责人: John Douglas Hulleman
• 金额: $ 18.86万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10657396
• 关键词: Adenoviruses; Biology; Cell Culture Techniques; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Core Grant; Custom; DNA; DNA Maintenance; Development; Documentation; Endotoxins; Future; Generations; Genetic Engineering; Glycerol; In Vitro; Individual; Infrastructure; Institution; Lentivirus; Maintenance; Modification; Molecular Biology; Mutagenesis; Phenotype; Plasmids; Point Mutation; Preparation; Procedures; Production; Proteins; Publications; Research; Research Personnel; Resources; Scientist; Services; Site-Directed Mutagenesis; System; Techniques; Testing; Transcript; Validation; Variant; Virus; Vision; Vision research; Western Blotting; Work; experimental study; imaging platform; immunocytochemistry; in vivo; insertion/deletion mutation; instrument; instrumentation; interest; knock-down; plasmid DNA; promoter; small hairpin RNA

Resource/Service Core: Molecular Biology/Virus Production Module Abstract: The Molecular Biology/Virus Production (MB/VP) Module will generate and validate DNA constructs primarily for use in mammalian cultures/systems in vision research. This module will also purify and concentrate both lentivirus and adenovirus for subsequent use in immortalized and primary cultures (maintained by individual investigators and the Cell Culture & Cell Phenotyping Module). The MB/VP Module will provide the following services: 1) De novo generation of constructs to be used for vision research 2) modification (mutagenesis, insertion/deletion) of plasmid DNA; 3) maintenance and propagation of DNA glycerol stocks; 4) production and purification of lentivirus and adenovirus; 5) validation of established DNA constructs and lentivirus/adenovirus by immunocytochemistry, qPCR and western blotting. The specific services provided within the MB/VP Module are: a) site-directed mutagenesis for generating point mutations, insertions, or deletion variants. b) custom Gibson assembly for development of new DNA fusion constructs driven by desired promoters of interest. c) shRNA and CRISPR/SpCas9 plasmid generation for knockdown/out experiments. d) full sequencing of generated constructs, extensive documentation, and maintenance of DNA stocks for future use. e) small to large scale endotoxin free plasmid purification for downstream applications. f) thorough confirmation of produced constructs at the transcript, protein and subcellular level. g) generation of high-purity lentivirus and adenovirus for use in vitro or in vivo. Overall, the MB/VP Module will support a range of enabling recombinant DNA technologies and provide the assistance and expertise required to test unique hypotheses posited by vision scientists at UTSW and neighboring institutions.

资源/服务核心：分子生物学/病毒生产模块 抽象的： 分子生物学/病毒生产 (MB/VP) 模块将主要生成和验证 DNA 构建体 用于视觉研究中的哺乳动物培养/系统。该模块还将纯化和浓缩两者 慢病毒和腺病毒随后用于永生化和原代培养物（由个人维护） 研究人员和细胞培养和细胞表型模块）。 MB/VP 模块将提供以下内容 服务：1）从头生成用于视觉研究的构建体2）修改（诱变， 质粒DNA的插入/删除； 3) DNA甘油原液的维持和繁殖； 4）生产和 慢病毒和腺病毒的纯化； 5) 已建立的DNA构建体和慢病毒/腺病毒的验证 通过免疫细胞化学、qPCR 和蛋白质印迹。 MB/VP 模块内提供的具体服务是： a) 用于产生点突变、插入或缺失变体的定点诱变。 b) 定制 Gibson 组装，用于开发由所需驱动的新 DNA 融合构建体 利益的推动者。 c) 用于敲除/敲除实验的 shRNA 和 CRISPR/SpCas9 质粒生成。 d) 对生成的构建体进行全面测序、广泛的记录以及 DNA 库存的维护 以供将来使用。 e) 小规模到大规模的无内毒素质粒纯化，用于下游应用。 f) 在转录本、蛋白质和亚细胞水平上彻底确认所产生的构建体。 g) 产生用于体外或体内的高纯度慢病毒和腺病毒。 总体而言，MB/VP 模块将支持一系列重组 DNA 技术，并提供 测试 UTSW 视觉科学家提出的独特假设所需的帮助和专业知识 邻近机构。