Screening Core

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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.