Screening Core

生物医学的最新进展使基于细胞的基因筛查成为可能 哺乳动物细胞以高通量方式发现基因特定功能。其中包括 人类和小鼠基因组的测序，非冗余全基因组集合的组装 CDNA，哺乳动物细胞中RNA干扰方法的发展，以及全基因组的组装 可以针对已知基因的短发夹状RNA(ShRNAs)的集合。该计划的总体目标 基因组资源核心是促进这些进展在该计划的四个项目中的应用，以 使询问T细胞受体(TCR)过程中的基因功能成为可能。 发信号。基因组资源核心将允许该计划中的研究人员访问cDNA和shRNA 有基因组覆盖并在约翰霍普金斯高中存储和维护的图书馆 吞吐量生物学(HIT)中心。使用基因组资源核心--波美兰茨实验室的设施 将提供一个集中设施来处理HIT中心提供的细菌等分，包括A) 从细菌基因组中提取DNA用于瞬时转染；B)包装表达shRNA 病毒构建成病毒，用于感染靶细胞；C)用包装病毒感染靶细胞；D) 将文库克隆cDNA插入适当的表达载体进行筛选；以及E)汇集 文库克隆，以最大限度地增加每次检测要筛选的基因数量。基因组资源的核心 将允许对在TCR参与和信号传递中起作用的基因进行无偏见筛选，以及 对假定在这些过程中起关键作用的候选基因进行有针对性的研究。项目1 将使用核心来识别在激活过程中参与TCR聚类的基因。项目3将使用核心来 测试候选基因在芽孢素介导的TCR信号调节中的作用。项目4将 使用Core研究酪氨酸激酶基因在TCR诱导的钙信号转导中的潜在作用。项目5 将使用核心来识别TCR信号到NF-KB的调制器。因为不适当TCR信令可能 导致无效的免疫监视、自身免疫或癌症，这些筛查工作的结果可能 为治疗免疫系统疾病的新疗法提供分子靶点。

Recent advances in biomedical science have made it possible to perform cell-based genetic screens in mammalian cells to uncover gene-specific functions in a high throughput fashion. These include the sequencing of the human and mouse genomes, the assembly of non-redundant genome-wide collections of cDNAs, the development of RNA interference methods in mammalian cells, and the assembly of genomewide collections of short hairpin RNAs (shRNAs) that can target known genes. The overall goal of the Genomic Resources Core is to facilitate the application of these advances in four projects of the Program, to make it possible to interrogate gene function during T cell receptor (TCR) engagement.and subsequent signaling. The Genomic Resources Core will grant investigators in the program access to cDNA and shRNA libraries that have genomic coverage and which are stored and maintained in the Johns Hopkins High Throughput Biology (HiT) Center. Using facilities in the Pomerantz laboratory, the Genomic Resources Core will provide a centralized facility for processing the bacterial aliquots provided by the HiT Center, including A) preparation of DNA from the bacterial aliquots for transient transfection; B) packaging of shRNA-expressing viral constructs into virus for infection of target cells; C) infection of target cells with packaged virus; D) the transfer of library clone cDNA inserts into appropriate expression vectors for screening; and E) pooling of library clones to maximize the number of genes to be screened per assay. The Genomic Resources Core will allow for unbiased screens for genes that play a role in TCR engagement and signaling, as well as for targeted study of candidate genes that are hypothesized to play critical roles in these processes. Project 1 will use the Core to identify genes involved in TCR clustering during activation. Project 3 will use the Core to test the role of candidate genes involved in the Sproutyl -mediated regulation of TCR signaling. Project 4 will use the Core to survey tyrosine kinase genes for potential roles in TCR-induced calcium signaling. Project 5 will use the Core to identify modulators of TCR signaling to NF-KB. Since inappropriate TCR signaling can result in ineffective immune surveillance, autoimmunity, or cancer, the results of these screening efforts may provide molecular targets for new therapies designed to treat diseases of the immune system.