Screening Core

生物医学科学的最新进展使得有可能进行基于细胞的遗传筛选， 哺乳动物细胞，以高通量方式揭示基因特异性功能。其中包括 人类和小鼠基因组的测序，非冗余的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， 短发夹RNA（shRNAs）的集合，可以靶向已知基因。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， 使得在T细胞受体（TCR）接合期间询问基因功能成为可能。 发信号。基因组资源核心将授予该计划的研究人员获得cDNA和shRNA的权利 具有基因组覆盖率并且在约翰霍普金斯高中储存和维护的文库 生物学（HiT）中心。利用Pomerantz实验室的设施，基因组资源核心 将提供一个集中设施，用于处理HiT中心提供的细菌等分试样，包括A） 从细菌等分试样中制备用于瞬时转染的DNA; B）包装表达shRNA的 将病毒构建体装入病毒中用于感染靶细胞; C）用包装的病毒感染靶细胞; D） 将文库克隆cDNA插入物转移到合适的表达载体中用于筛选;和E）合并 文库克隆以使每次测定待筛选的基因的数目最大化。基因组资源核心 将允许对在TCR接合和信号传导中起作用的基因进行无偏筛选，以及对 有针对性地研究候选基因，这些基因被假设在这些过程中发挥关键作用。项目1 将使用核心来识别在活化过程中参与TCR聚集的基因。项目3将使用核心 测试参与Sproutyl介导的TCR信号调节的候选基因的作用。项目4将 使用Core调查酪氨酸激酶基因在TCR诱导的钙信号传导中的潜在作用。项目5 将使用核心来鉴定TCR信号传导至NF-κ B的调节剂。由于不适当的TCR信号传导可以 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.