Tissue Culture Core - Core A

组织培养核心 - 核心 A

• 批准号: 10183286
• 负责人: JOSEPH L GOLDSTEIN
• 金额: $ 99.3万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10183286
• 关键词: Agarose Chromatography; Aliquot; Antibodies; Area; Baculoviruses; Biopsy; Cell Culture Techniques; Cell Line; Cells; Charge; Cholesterol Homeostasis; Clone Cells; Complementary DNA; Core Facility; Cultured Cells; Doctor of Medicine; Enhancers; Fibroblasts; Freezing; Generations; Genes; Growth; Head; Human; Hybridomas; Incubators; Injections; Insecta; Lentivirus; Liquid substance; Liver; Maintenance; Metabolic Diseases; Microscope; Molecular; Monoclonal Antibodies; Mus; Mutate; Nitrogen; Open Reading Frames; Oryctolagus cuniculus; Patients; Plasmids; Principal Investigator; Production; Program Research Project Grants; Proteins; Quality Control; Recombinant Proteins; Recombinant adeno-associated virus (rAAV); Recombinants; Research Personnel; Research Project Grants; Roller Bottle; Services; Skin; Staphylococcal protein A-sepharose; Sterility; Suspension Culture; Transfection; Work; cell preparation; experience; experimental study; gene function; in vivo; monoclonal antibody production; operation; permanent cell line; polyclonal antibody; promoter; protein purification; success; tissue culture

Core ATissue Culture Core will be responsible for providing investigators with cultured cells, monoclonal antibodies, recombinant proteins, and recombinant adeno-associated viruses. The Core will be directed by Dr. Joseph Goldstein, who has served as its Principal Investigator for the past 40 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 the past 28 years. The physical facilities of the Core consists of three suites of rooms that are used solely for tissue culture. The facility is equipped with 40 incubators, 14 inverted microscopes, 1 stereo microscope, 16 sterile work areas (hoods), 2 roller bottle apparatuses, 5 refrigerated incubator shakers, 3 table-top refrigerated centrifuges, 11 refrigerators, and 7 liquid nitrogen freezers for storage of cell lines. The success of the entire Program Project Grant depends on the smooth operation of this Core. Over the past 40 years, we have 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. Since 1984, tens of thousands of transfection experiments have been carried out in which we have introduced into various cell lines (e.g., HEK- 293, CHO, and SV589 cells) 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 A-Sepharose chromatography of more than 25 different polyclonal rabbit 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 insect cells in suspension culture for production of recombinant proteins by infecting these cells with recombinant baculoviruses encoding cloned cDNAs; 6) Production of recombinant adeno-associated viruses and lentiviruses, 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. C/PPG 2015 – Core A – 30-line Summary

Core A组织培养中心将负责向研究者提供培养的细胞， 单克隆抗体、重组蛋白和重组腺相关病毒。芯会 由Joseph Goldstein博士领导，他在过去的40年里一直担任首席研究员。的 核心的技术工作由五名经验丰富的技术人员进行，其中一名（丽莎贝蒂） 在过去的28年里负责这个设施核心的物理设施包括三个套房的房间 仅用于组织培养。该设施配备了40个培养箱，14个倒置显微镜，1个 体视显微镜，16个无菌工作区（通风橱），2个滚瓶装置，5个冷藏培养箱振荡器， 3台台式冷冻离心机、11台冰箱和7台用于储存细胞系的液氮冷冻机。 整个计划项目资助的成功取决于该核心的顺利运作。超过 在过去的40年里，我们在保持质量控制和增长方面积累了丰富的经验。 来自皮肤活检的多种细胞系，包括超过1100种不同的原代人成纤维细胞株 来自正常受试者以及来自代谢紊乱患者。自1984年以来， 已经进行了转染实验，其中我们已经引入各种细胞系（例如，HEK- 293、CHO和SV 589细胞）的多个质粒构建体，所述多个质粒构建体含有蛋白质编码区或 多个基因的启动子/增强子区域。从这些转染，超过3000稳定和永久的 细胞系已经克隆建立并以多个等分试样冷冻。 除了维持储备细胞系和制备用于实验的培养细胞外， 主要从事以下工作：1）小鼠和兔杂交瘤细胞系的建立和维持 和从培养基中产生单克隆抗体; 2）通过蛋白A-琼脂糖凝胶纯化 针对多种蛋白质的超过25种不同多克隆兔抗体的层析; 3） 大量悬浮培养细胞的生长，其允许cDNA的有效转染和生产 4）分离、维持和冷冻已被克隆的细胞系， 用各种cDNA和启动子/增强子构建体的突变形式转染; 5）维持 悬浮培养的昆虫细胞，用于通过感染这些细胞来生产重组蛋白 编码克隆cDNA的重组杆状病毒; 6）重组腺相关病毒的产生 以及慢病毒，用于评估培养细胞中基因的功能，以及植入后肝脏中基因的功能。 体内注射; 7）将单克隆和多克隆抗体和细胞系的等分试样发送到数百个 调查人员要求他们。 C/PPG 2015 -核心A - 30线总结