Tissue Culture & Antibody Production Core

组织培养

基本信息

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
项目类别：
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

项目摘要

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，单克隆抗体、重组蛋白和重组腺相关病毒。核心 1 将是由 Russell DeBose-Boyd 博士指导，Joseph Goldstein 博士（他曾指导过分子遗传学系组织培养设施40年）和梁国胜博士（在该部门工作了20年）。核心的技术工作由五位经验丰富的人完成技术人员，其中一位（丽莎·贝蒂）负责该设施已超过 30 年。物理方面核心设施包括四间仅用于组织培养的房间。设施配备配有 46 个培养箱、17 个倒置显微镜、1 个体视显微镜、16 个无菌工作区（罩）、2 个滚瓶设备、7 台冷藏培养箱摇床、3 台台式冷冻离心机、11 台冰箱和 7 台液体用于储存细胞系的氮气冷冻柜。整个计划项目补助金（PPG）的顺利完成取决于顺利运行核心 1. 核心在维持质量控制和发展多种产品方面积累了丰富的经验细胞系，包括 1100 多种不同的原代人成纤维细胞株，源自皮肤活检正常受试者以及先前 PPG 支持的代谢紊乱患者。数十个已在 Core 中进行了数千次转染实验，其中各种细胞系（例如 HEK- 293、CHO 和 SV589 细胞）已用含有以下任一者的多个质粒构建体转染蛋白质编码区或多基因的启动子/增强子区。从这些转染中，超过 3000 个稳定且永久的细胞系已克隆建立并分多份冷冻。除了维持储存细胞系和准备实验用培养细胞外，核心参与以下活动： 1) 小鼠和兔杂交瘤细胞系的生成和维护以及从培养基中生产单克隆抗体； 2) 通过 Protein G- 和 Protein A- 进行纯化针对小鼠/兔单克隆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，可有效转染 cDNA 及其编码蛋白质的生产； 4) 分离、维持和冷冻克隆细胞系已被各种 cDNA 和启动子/增强子构建体的突变版本转染； 5）维持哺乳动物和昆虫细胞的悬浮培养以生产重组蛋白用编码克隆 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，其用于评估培养细胞和体内注射后肝脏中的基因； 7) 发送单克隆和多克隆的等分试样抗体和细胞系提供给数百名需要的研究人员。