Genetics

遗传学

• 批准号: 7663285
• 负责人: YOSEF REFAELI
• 金额: $ 15.36万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7663285
• 关键词: AN-1; Antigen Receptors; Apoptosis Regulator; B-Lymphocytes; Cell Line; Cells; Development; Emerging Technologies; Family; Gene Expression; Generations; Genes; Genetic; Goals; Hematopoietic; Hematopoietic stem cells; Homeostasis; Lymphocyte; Lymphoid Cell; Mediating; Molecular; Mouse Strains; Mus; NF-kappa B; Nature; PTEN gene; Pathway interactions; Process; Production; Protein Family; Protein Overexpression; Receptors, Antigen, B-Cell; Research Personnel; Resources; Role; Signal Transduction; Specificity; Technology; Training; Training Support; Transgenic Mice; Viral; Viral Vector; autoreactive B cell; base; cDNA Expression; design; gain of function; genetic manipulation; improved; interest; killings; loss of function; member; new technology; pluripotency; programs; small hairpin RNA; tool; vector

This core aims to provide access, support and training for three new and emerging technologies for the projects described in this program. In addition, the core seeks to continue to develop and improve of the application of the three technologies described for the specific needs of the projects described in this program. The specific technologies include: a) the development of a comprehensive and technically advanced resource for the design, generation and production of viral-based shRNA vectors as well as retroviral approaches to manipulate ectopic gene expression in primary cells and cell lines of interest, b) the use of genetically modified mice for the efficient immortalization of autoreactive B-cells, and c) the use of novel technologies to conditionally immortalize long term hematopoietic stem cells in order to generate cell lines from transgenic mice of interest for these projects. The three specific aims of this core are: To develop a suite of viral vectors that will be useful for genetic manipulation of primary lymphoid cells. The use of viral mediated delivery of shRNA will be the primary tool for loss-of-function studies whereas the use of retroviral driven cDNA expression will be the primary means of gain-of-function studies in mice. 1. To use mice that overexpress MYC in an inducible, and B-cell specific manner to immortalize autoreactive B-cells (AN-1 cells). This approach will be important for Dr. Cambier's project and their attempts to determine the nature and specificity of the B-cell antigen receptors on normal AN1/T3 cells. 2. To define the hematopoietic pluripotency of conditionally immortalized long-term hematopoietic stem cell clones. The genetic pathways we will focus on will be of broad interest and application to the other projects in this program, and we will train and assist those investigators in this process as needed. This core will be important in allowing the different projects described in this program to define the molecular basis by which antigen receptor derived signals are able to regulate lymphocyte tolerance, homeostasis and survival. Depending on the specific circumstances, the antigen receptor can anergize, tolerize or kill lymphoid cells with the ultimate goal of generating an innocuous but useful repertoire. The projects described in this project will focus initially on the role of members of the Bcl-2 family in these functions of the antigen receptors.

该核心旨在为三种新兴技术提供访问、支持和培训 本计划中描述的项目。此外，核心力求继续发展和改进 为满足本文所述项目的具体需求而应用所描述的三种技术 程序。具体技术包括： a) 开发综合性技术 用于设计、生成和生产基于病毒的 shRNA 载体的先进资源以及 逆转录病毒方法操纵原代细胞和感兴趣的细胞系中的异位基因表达，b) 使用转基因小鼠实现自身反应性 B 细胞的有效永生化，以及 c) 使用 有条件地长期永生化造血干细胞以产生细胞的新技术 来自这些项目感兴趣的转基因小鼠的品系。该核心的三个具体目标是： 开发一套可用于原代淋巴细胞基因操作的病毒载体。这 使用病毒介导的 shRNA 递送将成为功能丧失研究的主要工具，而使用 逆转录病毒驱动的 cDNA 表达的研究将是小鼠功能获得研究的主要手段。 1. 使用以可诱导的、B细胞特异性方式过度表达MYC的小鼠来永生化 自身反应性 B 细胞（AN-1 细胞）。这种方法对于坎比尔博士的项目及其 尝试确定正常 AN1/T3 上 B 细胞抗原受体的性质和特异性 细胞。 2. 条件永生化长期造血干的造血多能性的定义 细胞克隆。 我们将重点关注的遗传途径将引起广泛的兴趣并应用于本研究中的其他项目 计划，我们将根据需要在此过程中培训和协助这些调查人员。这个核心将是 重要的是允许本计划中描述的不同项目定义分子基础 抗原受体衍生的信号能够调节淋巴细胞耐受性、稳态和存活。 根据具体情况，抗原受体可以激活、耐受或杀死淋巴细胞 最终目标是产生无害但有用的曲目。本项目中描述的项目 首先将重点关注 Bcl-2 家族成员在抗原受体的这些功能中的作用。