Tissue Culture Core - Core A

核心A组织培养核心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。核心将是 导演约瑟夫·戈尔茨坦博士，他在过去的40年里一直担任该组织的首席调查员。这个 核心的技术工作由五名经验丰富的技术人员执行，其中一名(丽莎·比蒂饰) 在过去的28年里负责这个设施。核心的物质设施由三套房间组成 仅用于组织培养。该设施配备了40个孵化器，14个倒置显微镜，1 立体显微镜，16个无菌工作区(罩)，2个滚筒设备，5个冷藏保温箱摇壶， 3台台式冷藏离心机、11台冰箱和7台液氮冷冻机，用于储存细胞系。 整个计划项目资助的成功与否取决于这一核心的顺利运行。完毕 在过去的40年里，我们在保持质量控制和发展壮大方面积累了丰富的经验。 多个细胞系，包括来自皮肤活检的1100多个不同的原代人类成纤维细胞株 来自正常受试者和代谢紊乱患者。自1984年以来，数以万计的 我们已经进行了转基因实验，其中我们已经将其导入了各种细胞系(例如，HEK- 293、CHO和SV589细胞)多个含有蛋白质编码区或 多基因启动子/增强子区域。从这些转染物中，有3000多个稳定和永久 已经克隆建立了细胞系，并将其冻结在多个等份中。 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A-琼脂糖凝胶纯化 针对多种蛋白质的超过25种不同的多克隆兔抗体的层析；3) 大量悬浮培养细胞的生长，使cDNA能够有效地转导和生产 4)分离、维护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版本转染；5)维持 悬浮培养的昆虫细胞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)重组腺相关病毒的生产 慢病毒，用来评估培养细胞和肝脏中基因的功能。 体内注射；7)将等量的单抗和多克隆抗体和细胞系发送到数百个 提出要求的调查人员。 C/PPG 2015-核心A-30行摘要

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