Tissue Culture & Antibody Production Core

组织培养

• 批准号: 10332594
• 负责人: Russell Alfred DeBose-Boyd
• 金额: $ 36.08万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10332594
• 关键词: Agarose Chromatography; Aliquot; Animals; Antibody Formation; Area; Baculoviruses; Biopsy; CRISPR/Cas technology; Cell Line; Cells; Charge; Clone Cells; Complementary DNA; Core Facility; Cultured Cells; Dependovirus; Enhancers; Fibroblasts; Freezing; GTP-Binding Proteins; Generations; Genes; Goals; Growth; Guidelines; Human; Hybridomas; Incubators; Injections; Insecta; Liquid substance; Liver; Maintenance; Mammalian Cell; Metabolic Diseases; Methods; Microscope; Molecular Genetics; Monoclonal Antibodies; Mus; Mutate; Nitrogen; Open Reading Frames; Oryctolagus cuniculus; Patients; Plasmids; Production; Program Research Project Grants; Proteins; Quality Control; Reagent; Recombinant DNA Research; Recombinant Proteins; Recombinant adeno-associated virus (rAAV); Recombinants; Research Personnel; Research Project Grants; Residual state; Roller Bottle; Skin; Staphylococcal protein A-sepharose; Sterility; Suspension Culture; Transfection; United States National Institutes of Health; Work; cardiovascular risk factor; cell preparation; experience; experimental study; gene function; genetic manipulation; in vivo; interest; lipid metabolism; monoclonal antibody production; novel strategies; operation; permanent cell line; polyclonal antibody; promoter; protein function; protein purification; tissue culture; tool

The Tissue Culture Core (Core 1) will be responsible for providing investigators with cultured cells, monoclonal antibodies, recombinant proteins, and recombinant adeno-associated viruses. Core 1 will be directed by Dr. Russell DeBose-Boyd, with the assistance of Dr. Joseph Goldstein (who has directed the Department of Molecular Genetics Tissue Culture Facility for the past 40 years) and Dr. Guosheng Liang (who has worked in the Department for 20 years). The technical work in the Core is carried out by five experienced technicians, one of whom (Lisa Beatty) has been in charge of this facility for more than 30 years. The physical facilities of the Core consist of four suites of rooms that are used solely for tissue culture. The facility is equipped with 46 incubators, 17 inverted microscopes, 1 stereo microscope, 16 sterile work areas (hoods), 2 roller bottle apparatuses, 7 refrigerated incubator shakers, 3 table-top refrigerated centrifuges, 11 refrigerators, and 7 liquid nitrogen freezers for storage of cell lines. The successful completion of this entire Program Project Grant (PPG) depends on the smooth operation of Core 1. The Core developed considerable experience in maintaining quality control and in growing multiple cell lines, including over 1100 different primary human fibroblast cell strains, derived from skin biopsies from normal subjects as well as from patients with metabolic disorders supported by previous PPGs. Tens of thousands of transfection experiments have been carried out in the Core in which various cell lines (e.g., HEK- 293, CHO, and SV589 cells) have been transfected with multiple plasmid constructs containing either the protein-coding region or the promoter/enhancer region of multiple genes. From these transfections, more than 3000 stable and permanent cell lines have been clonally established and frozen away in multiple aliquots. In addition to maintenance of stock cell lines and preparation of cultured cells for experiments, the Core is involved in the following activities: 1) Generation and maintenance of mouse and rabbit hybridoma cell lines and production of monoclonal antibodies from culture medium; 2) Purification by Protein G- and Protein A- Sepharose chromatography of mouse/rabbit monoclonal and rabbit polyclonal antibodies directed against multiple proteins; 3) Growth of large volumes of suspension-culture cells that allow efficient transfection of cDNAs and production of their encoded proteins; 4) Isolating, maintaining, and freezing away cloned cell lines that have been transfected with mutated versions of various cDNA and promoter/enhancer constructs; 5) Maintenance of mammalian and insect cells in suspension culture for production of recombinant proteins by infecting these cells with recombinant baculoviruses encoding cloned cDNAs; 6) Maintenance and production of cells for generation of recombinant adeno-associated viruses, which are used for evaluating the function of genes in cultured cells and in the liver after in vivo injection; 7) Sending aliquots of monoclonal and polyclonal antibodies and cell lines to hundreds of investigators who request them.

组织培养核心(核心1)将负责为研究人员提供培养细胞， 单抗、重组蛋白和重组腺相关病毒。核心1将是 由Russell DeBose-Boyd博士执导，Joseph Goldstein博士(曾执导 分子遗传学系组织培养设施40年)和梁国生博士(世卫组织 在司法部工作了20年)。核心的技术工作由五名经验丰富的人执行 技术人员，其中一位(Lisa Beatty)已经负责这个设施30多年了。体能 核心的设施由四套房间组成，这些房间仅用于组织培养。设施配备齐全 有46个保温箱、17个倒置显微镜、1个立体显微镜、16个无菌工作区(罩)、2个滚筒 设备、7个冷藏孵化器摇床、3台台式冷藏离心机、11个冰箱和7个液体 用于储存细胞系的氮气冷冻机。 整个计划项目拨款(PPG)的顺利完成依赖于顺利运行 核心1.核心在维护质量控制和发展多项业务方面积累了丰富的经验 细胞系，包括1100多个不同的原代人类成纤维细胞株，来自于来自 正常受试者以及之前PPG支持的代谢紊乱患者。几十个 在核心中已经进行了数千次的转基因实验，其中各种细胞系(例如，HEK- 293、CHO和SV589细胞)已被多个含有 蛋白质编码区或多基因的启动子/增强子区域。从这些转基因中，超过 已经克隆建立了3000个稳定和永久的细胞系，并将其冷冻在多个等量中。 除了维持现有的细胞系和为实验准备培养的细胞外，核心 参与了以下活动：1)小鼠和兔杂交瘤细胞系的建立和维持 2)蛋白G-和蛋白A-纯化。 抗鼠/兔单抗和兔多克隆抗体的琼脂糖层析 多种蛋白质；3)大量悬浮培养细胞的生长，使其能够有效地转导 CDNA及其编码蛋白的产生；4)克隆细胞系的分离、维持和冷冻 已被多种cdna和启动子/增强子构建体突变的载体；5) 哺乳动物和昆虫细胞悬浮培养生产重组蛋白的维持 用编码克隆cDNA的重组杆状病毒感染这些细胞；6)维护和生产 用于产生重组腺相关病毒的细胞的数量，用于评估细胞的功能 体内注射后培养细胞和肝脏中的基因；7)发送等量的单克隆和多克隆 抗体和细胞系提供给数百名提出要求的研究人员。