Tissue Culture & Antibody Production Core

组织培养

基本信息

批准号：
10543866
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/10543866
关键词：
Agarose Chromatography Aliquot Animals Antibodies Antibody Formation Area Baculoviruses Biopsy CRISPR/Cas technology Cell Line Cells Charge Clone Cells Complementary DNA Core Facility Cultured Cells Dedications Dependovirus Enhancers Fibroblasts Freezing G-substrate 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 Refrigeration 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 stable cell line 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的细胞，单克隆抗体，重组蛋白和重组腺相关病毒。核心1将是由罗素·德·德·鲍尔（Russell Debose-Boyd）博士在约瑟夫·戈德斯坦（Joseph Goldstein）博士的协助下执导（他指导了在过去40年中，分子遗传学组织培养设施）和Guosheng Liang博士（谁在该部门工作了20年）。核心中的技术工作由五个经验丰富的技术人员，其中一位（Lisa Beatty）负责此设施已有30多年了。身体核心的设施由四个房间套件组成，这些房间仅用于组织培养。该设施配备了具有46个孵化器，17个倒置显微镜，1个立体显微镜，16个无菌工作区域（引擎盖），2个滚筒瓶设备，7种冷藏孵化器，3个台式冷藏离心机，11个冰箱和7个液体用于存储细胞系的氮冷冻器。整个计划项目赠款（PPG）的成功完成取决于平稳的操作核心1。核心在维持质量控制和增长多重方面具有丰富的经验细胞系，包括1100多种不同的原代人成纤维细胞菌株，这些细胞菌株来自皮肤活检。正常受试者以及先前PPG支持的代谢疾病患者的患者。数十个数以千计的转染实验是在各种细胞系（例如Hek-）的核心中进行的 293，CHO和SV589细胞）已用包含多个质粒构建体转染蛋白质编码区域或多个基因的启动子/增强子区域。从这些转染中，不仅仅是 3000个稳定和永久的细胞系已被克隆建立并在多个等分试样中冻结。除了维持库存细胞系和制备培养细胞进行实验之外，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产生单克隆抗体； 2）蛋白质和蛋白A-纯化针对针对的小鼠/兔单克隆和兔多克隆抗体的Sepharose色谱法多种蛋白质； 3）大量悬浮文化细胞的生长，可有效转染 cDNA和其编码蛋白的生产； 4）隔离，维持和冻结克隆的细胞系已被各种cDNA和启动子/增强子构建体的突变版本转染； 5）维持在悬浮培养中维持哺乳动物和昆虫细胞，以生产重组蛋白用编码克隆的cDNA的重组杆状病毒感染这些细胞； 6）维护和生产用于生成重组腺相关病毒的细胞的细胞，用于评估体内注射后培养细胞和肝脏中的基因； 7）发送单克隆和多克隆的等分试样抗体和细胞线的数百名调查人员要求。