Despite the recent completion of the human genome project, an ostensibly more difficult post-genomic challenge will be the functional annotation of all human genes and integration of this information into an operational cell-based model. Unfortunately, this is at present challenging, primarily due to the absence of reliable experimental and bioinformatic toolsets to rapidly delineate and describe gene function en masse. Insertional mutagenesis has proven to be an extremely potent and versatile experimental tool to knock-out expression of targeted genes, allowing the development of a loss-of-function genetic screen platform based on mouse embryonic stem cells. Despite these successes, high-throughput (HT) genome-wide mutagenesis screening is technically challenging in somatic cells and significant limitations in the technology exist. Moreover, the technology is limited to very stringent positive selections, which greatly limits the number of biological applications. To address these shortcomings, we propose to develop a novel experimental platform to identify cancer-specific modulator genes on a genome-wide scale. The ultimate goal of the proposed project is to develop Array-assisted Insertional Mutagenesis (AIM) technology and apply this technology for genome-wide discovery and public domain database development of genes modulating drug responses in a wide range of cancer cell lines. Under Phase I support, we propose to establish and validate a universal AIM gene-discovery platform that would enable unequivocal genome-wide functional identification of cancer-related genes using either a positive or negative selection in conditions of either low or high stringency. No equivalent platform currently exists and the work, if successful, would enable the delineation of drug response mechanisms in cancer cells, and the application of this knowledge to translational research programs. In collaboration with our biology consultants from Cleveland Clinic, we will validate and apply AIM technology to uncover the determinants of Taxol response in human prostate cancer cells. As a result of the Phase I program, the AIM lentiviral vectors and supporting products will be released as ready-to-use commercial reagents for genome-wide functional analysis. Forward genetic screens with AIM technology have the potential to greatly simplify validation of gene functions and significantly impact the molecular dissection of cancer disease mechanisms.
AIM screens harbor considerable promise to identify new cancer drug targets for therapeutic intervention, and develop increasingly relevant paradigms for drug discovery. As a result, we foresee that these toolsets will significantly improve the efficiency, economy, and ease of performing HT genetic screens, and will provide basic researchers with preferred, cost-effective alternatives to existing commercially available reagents.
| Singhal, Ruchi; Deng, Xiaotao; Chenchik, Alex A et al. (2011) Long-distance effects of insertional mutagenesis. PLoS One 6:e15832 |
