Core--Gene knockout cell line

核心--基因敲除细胞系

• 批准号: 6563941
• 负责人: David A Sassoon
• 金额: $ 23.8万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6563941
• 关键词: animal breeding; biomedical facility; cell line; gene targeting; genetically modified animals; laboratory mouse; tissue /cell culture

DESCRIPTION: (Applicant's Description) The purpose of the Gene Knockout Cell Line Core is to collect, produce, and propagate cell lines derived from various gene knockout mice, since utilizing various null cell lines is central to all the projects proposed in this application. Among cells that this facility will generate and maintain are p53-/-, p53/mdm2-/-, Rb-/-, p21-/-, JNK-/-, and BRCA1-/- cells. Additional cultures from KO mice will be sought, as they become available. The core is directed by Drs. Manfredi (5 percent) and Sassoon (5 percent). A Research Assistant (100 percent effort) with a strong background in tissue culture technique (Amy Ream) will be responsible for the daily operations of this Core. Among the functions shared between all program project participants include the following: The Core will be responsible for purchasing and collecting various cell lines derived from gene knockout mice if they are available. For null cells that are not available as a cell line, the Core will purchase or collect the knockout mice. The mutant mice will be bred to homozygosity. Embryonic fibroblast cell lines (monitored via PCR) cultures will be generated and provided to the respective scientists in a timely manner. Dr. Sassoon has extensive experience with knockouts. For the preparation of MEF from embryos Dr. Douglas Forrest of the Department of Genetics at MSSM will advise the Core facility. Dr. Forrest is experienced in these protocols and is currently collaborating with several investigators in our cancer center (letter enclosed). Attempts to generate immortalized cultures will be made. The Core?s budget includes funds to house and breed various knockout mice at the Mount Sinai Animal facility, as well as for the day to day maintenance indicated above for purpose of generating cell lines. The KO derived cell lines will be stored at the Derald Ruttenberg Cancer Center cryogen storage facility, which is part of DHRCC service to MSSM community. The core will be responsible for collecting and maintaining hybridoma cell lines to generate monoclonal antibodies commonly used in the projects, such as monoclonal antibodies against p53, mdm2, p21, and BRCA1. In addition, the core will serve as a source for normalized growth of frequently used cell lines at earliest possible passages, such as 293, 293T, CV-1, cos-7, saos-2, (10)1, and NIH3T3. Technology and DNA constructs for generating tet regulatable cell lines have been transferred from Project 1 to the Core. This allows the Core to establish tet regulatable cell lines for genes of specific interest to different projects. The Core will provide quality control for identifying proper batches of Fetal Bovine Serum used by Program Project scientists. Support by the Core of specific projects will be determined during the monthly meetings of investigators. Priority will be based on the interdisciplinary scope of the requests as well as scientific merit in advancing important aims within individual projects.

描述：（申请人的描述）基因基因敲除单元的目的 线核是收集，产生和传播从 各种基因敲除小鼠，因为使用各种无效细胞系是中心的 对于本应用程序中提出的所有项目。在这个细胞中 设施将生成和维护为p53 - / - ，p53/mdm2 - / - ，rb - / - ，p21 - / - ， JNK - / - 和BRCA1 - / - 细胞。将寻求来自KO小鼠的其他文化， 随着它们的可用。核心由Drs执导。曼弗雷迪（5％） 和沙通（5％）。研究助理（100％的努力） 组织培养技术（Amy Ream）的强大背景将负责 对于此核心的日常操作。在所有之间共享的功能中 计划项目参与者包括以下内容：核心将是 负责购买和收集来自基因的各种细胞系 敲除老鼠如果可用。对于不可用的零单元 细胞系，核心将购买或收集淘汰小鼠。突变体 小鼠将繁殖到纯合性。胚胎成纤维细胞细胞系（监测 通过PCR）将生成文化并提供给各自科学家 及时。 Sassoon博士在淘汰赛方面拥有丰富的经验。为了 从胚胎的MEF准备MEF，Douglas Forrest博士 MSSM的遗传学将为核心设施提供建议。 Forrest博士经验丰富 这些协议，目前正在与几位调查人员合作 我们的癌症中心（封闭字母）。试图产生不朽的 将建立文化。 核心预算包括用于房屋和繁殖各种淘汰小鼠的资金 西奈山动物设施以及日常维护 上面指示的目的是生成细胞系。 KO衍生的细胞 线将存储在Derald Ruttenberg癌症中心的低温储存 设施，是MSSM社区DHRCC服务的一部分。核心将是 负责收集和维持杂交瘤细胞系以生成 项目中常用的单克隆抗体，例如单克隆 抗P53，MDM2，P21和BRCA1的抗体。此外，核心将 充当经常使用的细胞系在 最早的段落，例如293，293t，cv-1，cos-7，saos-2，（10）1，和 NIH3T3。用于生成TET可调节细胞的技术和DNA构建体 线已从项目1转移到核心。这允许核心 建立针对特定兴趣基因的TET可调节细胞系 不同的项目。核心将提供质量控制以识别 项目科学家使用的适当批次胎牛血清。 特定项目核心的支持将在月度期间确定 调查人员的会议。优先级将基于跨学科 请求的范围以及促进重要目标的科学优点 在各个项目中。