Tissue Culture & Antibody Production Core

组织培养

• 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
• 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

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）将负责向研究者提供培养细胞， 单克隆抗体、重组蛋白和重组腺相关病毒。核心1将是 导演罗素DeBose-Boyd博士，在约瑟夫·戈尔茨坦博士的协助下（谁曾指导过 在过去40年中，分子遗传学系组织培养设施）和梁国胜博士（ 他在这个部门工作了20年）。核心的技术工作由五位经验丰富的 技术人员，其中一人（丽莎贝蒂）已负责这一设施超过30年。物理 核心的设施包括四套房间，仅用于组织培养。该设施配备了 配备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（防护罩）、2个滚瓶 设备，7个冷冻孵育摇床，3个台式冷冻离心机，11个冰箱和7个液体 用于储存细胞系的氮气冷冻器。 整个计划项目补助金（PPG）的顺利完成取决于 核心1。核心开发了相当多的经验，在保持质量控制和增长多个 细胞系，包括超过1100种不同的原代人成纤维细胞株，来源于皮肤活检， 正常受试者以及既往PPG支持的代谢紊乱患者。数千 已经在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SV 589细胞）转染了多个质粒构建体，所述质粒构建体含有 蛋白质编码区或多个基因的启动子/增强子区。通过这些转染， 已经克隆建立了3000个稳定和永久的细胞系，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G-和蛋白A-纯化 小鼠/兔单克隆抗体和兔多克隆抗体的琼脂糖色谱法 3）生长大体积的悬浮培养细胞，其允许高效转染多种蛋白质; cDNA及其编码蛋白的生产; 4）分离、维持和冷冻掉克隆细胞系 已经用各种cDNA和启动子/增强子构建体的突变形式转染; 5） 维持悬浮培养中的哺乳动物和昆虫细胞用于通过以下方法生产重组蛋白： 用编码克隆cDNA的重组杆状病毒感染这些细胞; 6）维持和生产 用于产生重组腺相关病毒的细胞，其用于评估 体内注射后培养的细胞和肝脏中的基因; 7）将等份的单克隆和多克隆抗体 抗体和细胞系提供给数百名需要它们的研究人员。