Tissue Culture Laboratory

组织培养实验室

• 批准号: 7217726
• 负责人: JOSEPH L GOLDSTEIN
• 金额: $ 105.91万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217726
• 关键词: 3-hydroxy-3-methylglutaryl-coenzyme A; Adenoviruses; Aliquot; American Type Culture Collection; Amphotericin; Antibodies; Area; Baculoviruses; Biochemical; Biological; Biological Assay; Biopsy; Boston; COS Cells; Carbon Dioxide; Cell Line; Cells; Chinese Hamster Ovary Cell; Cholesterol; Cholesterol 7-alpha-Monooxygenase; Chromatography; Clone Cells; Cloning; Collection; Column Chromatography; Complementary DNA; Condition; Core Facility; Count; Culture Media; Cultured Cells; Data; Diploidy; Drosophila genus; Embryo; Endopeptidases; Ensure; Equipment; Evaluation; Familial Hypercholesterolemia; Fibroblasts; Freezing; G-substrate; Gene Targeting; Generations; Genes; Growth; Hela Cells; Human; Hybridomas; Hydroxymethylglutaryl-CoA reductase; Immunoglobulin G; Incubators; Insecta; Kidney; Laboratories; Laboratory Assistant; Learning; Liquid substance; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Maintenance; Marshal; Measures; Membrane; Metabolic; Metabolic Diseases; Metabolism; Methods; Microscope; Microscopic; Mixed Function Oxygenases; Molecular Genetics; Monitor; Monoclonal Antibodies; Multiple Myeloma; Mus; Mutate; Mycoplasma; Neoplasms; Nitrogen; Numbers; Open Reading Frames; Operative Surgical Procedures; Oryctolagus cuniculus; Patients; Peptide Hydrolases; Phase; Plasmids; Polymerase Chain Reaction; Preparation; Procedures; Production; Program Research Project Grants; Promoter Regions; Property; Proteins; Quality Control; Rab geranylgeranyltransferase; Rat-1; Rate; Rattus; Research Personnel; Research Project Grants; Resistance; Roller Bottle; Rosa; SR-BI receptor; SRE-1 binding protein; Safety; Sampling; Serum; Services; Side; Site; Skin; Somatic Cell; Standards of Weights and Measures; Sterility; Suspension Culture; Testing; Time; Transfection; Universities; VLDL receptor; Variant; Week; Work; apolipoprotein E receptor 2; base; cell type; day; design; embryonic stem cell; experience; flasks; fluorescence microscope; hepatoma cell; mutant; oxysterol binding protein; permanent cell line; programs; promoter; reconstitution; research study; senescence; site-1 protease; success; tissue culture; tissue/cell culture

The Tissue Culture Core Laboratory will be responsible for providing investigators with cultured cells, antibodies, and facilities for routine microscopic analysis of biological samples. The core will be directed by Dr. Goldstein. He will be assisted by Drs. Y.K. Ho, Richard Anderson, Joachim Herz, and Rob Rawson. The technical work in the Tissue Culture Facility of this Core is carried out by four experienced technicians (Lisa Beatty, Angela Carroll, Jill Fairless, Marissa Hodgin) and one laboratory assistant (Rose Marshall). The Tissue Culture Facility of this Core is located in the Department of Molecular Genetics and consists of three suites of rooms that are used solely for tissue culture. One suite contains four work modules that open into a common work area; the second suite contains three work modules that open into a common work area; and the third suite contains two work modules, one for adenovirus work and the other for baculovirus. Each module is equipped with a sterile work area (Biological Safety Cabinet hood), one or more CO2 incubators, a refrigerator, and an inverted microscope. The common work area in each of the suites contains one or two sterile work areas. The entire facility contains a total of 12 inverted microscopes (6 of which are phase-contrast microscopes), 1 stereo microscope, 15 sterile work areas (hoods), 28 singlechamber CO2 incubators, 2 non-CO2 refrigerated incubators, 1 roller bottle incubator, 3 refrigerated incubator shakers, 3 table-top refrigerated centrifuges, and 9 refrigerators. Four liquid nitrogen freezers for storage of cell lines are located in the common work area adjacent to the work modules. In addition to this standard equipment, we have a Zeiss Axiovert 35 M inverted fluorescence microscope that allows us to visualize cells directly in the Petri dish (under sterile conditions) so as to determine whether or not they have taken up reconstituted fluorescent LDL. This method is the assay of choice for identifying and monitoring mutant cholesterol auxotrophic cell clones that are resistant to amphotericin and thus fail to take up fluorescent LDL (Research Project 1). The success of the entire Program Project depends on the smooth operation of the Tissue Culture Facility. Each of the six Research Projects will depend directly on the availability of cultured cells. Over the past 33 years, we have developed considerable experience in maintaining and growing multiple cell types, including diploid human fibroblasts, Chinese hamster ovary (CHO) cells, Sp2/O-Ag14 myeloma cells, simian COS cells, human embryonic kidney (HEK)-293 cells, rat-1 fibroblasts, mouse embryonic fibroblasts, human lymphoblastoid cells, HeLa cells, rat FT02B hepatoma cells, human Huh-7 hepatoma cells, SV-40 transformed fibroblasts (SV-589), Sf9 insect cells, High Five insect cells, and Drosophila S2 cells. We have also become expert in culturing pluripotent mouse embryonic stem cells (ES cells), which will be used in Research Projects 2, 3, 5, and 6 for gene targeting experiments. Since 1973, over 1000 different primary human fibroblast cell strains, derived from skin biopsies from normal subjects as well as from patients with metabolic disorders, have been initiated in our laboratory. Multiple aliquots of cells from early passages of each of these strains are stored in liquid nitrogen. Our collection of frozen mutant cell lines includes fibroblasts from more than 160 subjects with the homozygous form of familial hypercholesterolemia (FH). Remarkably, we have been successful in carrying out metabolic experiments on mutant fibroblasts that we froze away 32 years ago. Since 1984, thousands of transfection experiments have been carried out in which we have introduced into various cell lines multiple plasmid constructs containing either the protein-coding region or the promoter regions of the genes for the LDL receptor, HMG CoA reductase, HMG CoA synthase, the oxysterol binding protein, SREBP-1 and -2, SCAP, Site-1 protease, Site-2 protease, lnsig-1 and -2, and.a and psubunits of farnesyltransferasel and a and ft-subunits of Rab geranylgeranyl transferase, REP-1 and -2, cholesterol 7-hydroxylase, oxysterol 7-hydroxylase, VLDL receptor, various domains of LRP, RAP, ApoER2, SR-B1 scavenger receptor, ARM, ABCG5/8, PCSK9, etc. From these transfections, more than 1200 stable and permanent cell lines have been established and used for biochemical analyses. Multiple aliquots of each of these transfected cell lines have been frozen in liquid nitrogen. These numbers do not include the thousands of transient transfections performed in HEK-293, COS, CHO cells, and SV589 cells. Based on current experience, we anticipate that we will be setting up for experiments approximately 600 Petri dishes of cells each day, of which 350 will be small dishes (60-mm) and 250 will be large dishes (100-mm). We have learned that certain aspects of quality control are essential in obtaining reproducible results for metabolic experiments in tissue culture cells. The following measures are rigorously enforced in our dayto- day operation: 1) An aliquot of calf serum from each batch of 100 liters is pretested prior to its general use so as to ensure that the serum is not toxic to our fibroblasts and that it supports a high cloning efficiency for CHO cells. 2) Cells in current use are routinely checked for Mycoplasma contamination in two ways: 1) by sending aliquots of the medium from 10 different cell lines to the ATCC Mycoplasma Testing Service (Manassas, VA) once per year; and 2) by our own testing with the use of a Myoplasma PCR Elisa test kit (Roche). 3) The generation time of each human fibroblast cell strain is monitored by a standard procedure, as follows: each week, each confluent stock flask is split 1:2, and the passage number is raised by 2. All stock flasks are discarded when the passage number exceeds 25. This procedure ensures that the growth rates of different aliquots of the same cell strain do not differ appreciably, and it also ensures that our experiments are carried out on cells that are well within Phase II of their growth cycle (Littlefield, J.W. In Variation, Senescence, and Neoplasia in Cultured Somatic Cells. Harvard University Press, Boston, 1976. pp. 54-77). 4) In setting up a typical experiment with nontransfected or permanently transfected cells, cells from the stock flasks are trypsinized under rigidly controlled conditions, counted using a hemacytometer, and 10 to 30 replicate dishes of cells are prepared under identical conditions. 5) In experiments designed to compare a normal and a mutant cell line or a nontransfected and a transfected cell line, Petri dishes of cells from both cell lines are always set up in parallel on the same day and grown in the same medium side by side in the same incubator. With these precautions, among others, our laboratory has been able to generate data on fibroblasts and other cell types that are reproducible from year to year, thus enabling rapid progress in the delineation of metabolic processes. In addition to the maintenance of stock cell lines and preparation of cultured cells for experiments, the Tissue Culture Core will be involved in the following activities: 1) Maintenance of our previously characterized hybridoma cell lines in suspension culture (in roller bottles) and production of large amounts of IgG fractions of monoclonal antibodies from culture medium. The following monoclonal antibodies are currently being produced and purified by Protein G column chromatography for use by investigators in the Program Project: anti-LDL receptor (lgG-C7); anti-SREBP-1 and anti-SREBP-2 (lgG-2A4, lgG-7D4, lgG-1C6, lgG-1D2); anti-Scap (lgG-9D5); anti-HMG CoA reductase (lgG-A9); and anti-Myc (lgG-9E10). Several new monoclonal antibodies against the membrane domain of Scap and against different domains of PCSK9 are currently being characterized. Moreover, mice have recently been injected with purified human lnsig-1 and lnsig-2 for producing hybridomas. 2) Purification by Protein G chromatography of more than 20 different polyclonal rabbit antibodies directed against the multiple proteins that are studied in this Program Project Grant. 3) Growth of large volumes of HEK-293S cells in suspension culture that allow the efficient transfection of cDNAs and the production of their encoded proteins. The HEK-293S cells have proved extremely valuable in the initial evaluation of the functional properties of proteins produced by newly cloned cDNAs. 4) Isolating, maintaining, and freezing away cloned cell lines that have been transfected with mutated versions of various cDNA and promoter constructs

组织培养核心实验室将负责为研究人员提供培养细胞， 抗体，以及用于生物样品常规显微分析的设施。核心将由 戈尔茨坦博士。他将得到博士的协助。 Y.K.何、理查德·安德森、约阿希姆·赫兹和罗布·罗森。 该核心组织培养设施的技术工作由四名经验丰富的人员负责 技术人员（Lisa Beatty、Angela Carroll、Jill Fairless、Marissa Hodgin）和一名实验室助理（Rose 马歇尔）。该核心的组织培养设施位于分子遗传学和 由三间仅用于组织培养的房间组成。一套套房包含四件作品 打开公共工作区的模块；第二个套件包含三个工作模块，这些模块打开为 公共工作区；第三个套件包含两个工作模块，一个用于腺病毒工作，另一个用于 杆状病毒。每个模块均设有无菌工作区（生物安全柜罩）、一个或多个 CO2 培养箱、冰箱和倒置显微镜。每间套房的公共工作区 包含一或两个无菌工作区。整个设施共有 12 台倒置显微镜（其中 6 台） 相差显微镜）、1 个体视显微镜、15 个无菌工作区（罩）、28 个单室 CO2 培养箱、2 个非 CO2 冷藏培养箱、1 个滚瓶培养箱、3 个冷藏培养箱 摇床、3 台台式冷冻离心机和 9 台冰箱。四个液氮冷冻柜用于储存 细胞系位于与工作模块相邻的公共工作区域中。除本标准外 设备，我们有一台 Zeiss Axiovert 35 M 倒置荧光显微镜，使我们能够观察细胞 直接放入培养皿中（在无菌条件下）以确定它们是否已吸收 重组荧光低密度脂蛋白。该方法是鉴定和监测突变体的首选测定方法 对两性霉素具有抗性的胆固醇营养缺陷型细胞克隆，因此无法吸收荧光 LDL （研究项目1）。 整个计划项目的成功取决于组织培养的顺利运行 设施。六个研究项目中的每一个都将直接取决于培养细胞的可用性。超过 过去 33 年，我们在维持和培养多种细胞类型方面积累了丰富的经验， 包括二倍体人成纤维细胞、中国仓鼠卵巢 (CHO) 细胞、Sp2/O-Ag14 骨髓瘤细胞、猿猴细胞 COS 细胞、人胚肾 (HEK)-293 细胞、rat-1 成纤维细胞、小鼠胚胎成纤维细胞、人 类淋巴母细胞、HeLa 细胞、大鼠 FT02B 肝癌细胞、人 Huh-7 肝癌细胞、SV-40 转化的成纤维细胞 (SV-589)、Sf9 昆虫细胞、High Five 昆虫细胞和果蝇 S2 细胞。我们有 也成为培养多能小鼠胚胎干细胞（ES细胞）的专家，该细胞将用于 研究项目 2、3、5 和 6 用于基因靶向实验。 自 1973 年以来，超过 1000 种不同的原代人类成纤维细胞株，源自皮肤活检 我们的实验室已开始对正常受试者以及患有代谢紊乱的患者进行研究。 将来自这些菌株中的每一种的早期传代的细胞的多个等分试样储存在液氮中。我们的 冷冻突变细胞系集合包括来自 160 多名纯合受试者的成纤维细胞 家族性高胆固醇血症（FH）的一种形式。值得注意的是，我们已经成功地进行了代谢 32 年前，我们对突变成纤维细胞进行了冷冻实验。 自 1984 年以来，我们已进行了数千次转染实验 将含有蛋白质编码区或 LDL 受体、HMG CoA 还原酶、HMG CoA 合酶、氧甾醇基因的启动子区域 结合蛋白、SREBP-1 和 -2、SCAP、Site-1 蛋白酶、Site-2 蛋白酶、lnsig-1 和 -2、and.a 和 p 亚基 法呢基转移酶和 Rab 香叶基香叶基转移酶的 a 和 ft 亚基、REP-1 和 -2， 胆固醇 7-羟化酶、氧甾醇 7-羟化酶、VLDL 受体、LRP、RAP、ApoER2 的各个结构域、 SR-B1清道夫受体、ARM、ABCG5/8、PCSK9等。从这些转染中，超过1200个稳定的 永久细胞系已经建立并用于生化分析。每份多份 这些转染的细胞系已冷冻在液氮中。这些数字不包括 在 HEK-293、COS、CHO 细胞和 SV589 细胞中进行了数千次瞬时转染。 根据目前的经验，我们预计我们将大约进行实验 每天 600 个细胞培养皿，其中 350 个为小培养皿（60 毫米），250 个为大培养皿 （100 毫米）。 我们了解到，质量控制的某些方面对于获得可重复的结果至关重要 用于组织培养细胞的代谢实验。我们日常严格执行以下措施： 日间操作： 1) 在一般使用前对每批 100 升小牛血清进行预测试，以便 确保血清对我们的成纤维细胞无毒，并且支持 CHO 的高克隆效率 细胞。 2) 目前使用的细胞通过两种方式常规检查支原体污染： 1) 通过 将 10 种不同细胞系的培养基等分试样发送至 ATCC 支原体检测服务 （弗吉尼亚州马纳萨斯）每年一次； 2) 通过我们自己使用支原体 PCR Elisa 检测试剂盒进行的检测 （罗氏）。 3)通过标准程序监测每个人成纤维细胞株的世代时间，如 如下：每周，每个汇合储备瓶按 1:2 分流，传代数增加 2。所有储备液 当传代次数超过 25 次时，烧瓶将被丢弃。此程序可确保培养皿的生长速率 同一细胞株的不同等分试样不会有明显差异，这也确保了我们的实验是 对处于生长周期第二阶段的细胞进行（Littlefield，J.W. In Variation， 培养体细胞的衰老和肿瘤形成。哈佛大学出版社，波士顿，1976 年。第 54-77 页）。 4) 在使用未转染或永久转染的细胞进行典型实验时，来自 将储备烧瓶在严格控制的条件下进行胰蛋白酶消化，使用血细胞计数器进行计数，并且10至 在相同条件下制备 30 个细胞重复培养皿。 5) 在旨在比较正常细胞系和突变细胞系或未转染细胞系和转染细胞系的实验中 转染的细胞系，来自两种细胞系的细胞的培养皿总是在同一天平行设置 并在同一培养箱中并排在同一培养基中生长。通过这些预防措施，除其他外， 我们的实验室已经能够生成可重复的成纤维细胞和其他细胞类型的数据 逐年进行，从而使代谢过程的描绘取得快速进展。 除了维持储存细胞系和准备实验用培养细胞外， 组织文化核心将参与以下活动： 1) 在悬浮培养中维持我们先前表征的杂交瘤细胞系（在滚筒中 瓶）以及从培养基中生产大量单克隆抗体的 IgG 部分。这 目前正在使用 Protein G 柱生产和纯化以下单克隆抗体 供项目研究人员使用的色谱法：抗 LDL 受体 (IgG-C7)；抗SREBP-1 和抗SREBP-2 (IgG-2A4、IgG-7D4、IgG-1C6、IgG-1D2)；抗 Scap (IgG-9D5)；抗 HMG CoA 还原酶 (lgG-A9)；和抗 Myc (IgG-9E10)。几种针对膜结构域的新单克隆抗体 目前正在对 Scap 和针对 PCSK9 不同域的特征进行表征。此外，小鼠还具有 最近注射了纯化的人 lnsig-1 和 lnsig-2 以产生杂交瘤。 2) 通过 Protein G 层析纯化 20 多种不同的多克隆兔抗体 针对本计划项目拨款中研究的多种蛋白质。 3) 悬浮培养中大量 HEK-293S 细胞的生长，可实现高效 cDNA 的转染及其编码蛋白的生产。 HEK-293S细胞已证明 对于新克隆产生的蛋白质的功能特性的初步评估非常有价值 cDNA。 4) 分离、维持和冷冻转染突变的克隆细胞系 各种 cDNA 和启动子构建体的版本