Array-assisted Insertional Mutagenesis Platform for Forward Genetics of Cancer

描述(申请人提供)：尽管最近完成了人类基因组计划，但一个表面上更困难的后基因组挑战将是所有人类基因的功能注释，并将这些信息整合到一个可操作的基于细胞的模型中。不幸的是，这在目前是具有挑战性的，主要是因为缺乏可靠的实验和生物信息学工具集来快速描述和描述整体的基因功能。插入突变已被证明是一种极其有效和通用的实验工具，可以敲除靶基因的表达，从而开发出基于小鼠胚胎干细胞的功能丧失的遗传筛选平台。尽管取得了这些成功，但在体细胞中进行高通量(HT)全基因组突变筛选在技术上是具有挑战性的，而且该技术存在重大限制。此外，该技术仅限于非常严格的积极选择，这极大地限制了生物应用的数量。为了解决这些缺点，我们建议开发一种新的实验平台，在全基因组范围内识别癌症特异性调控基因。 该项目的最终目标是开发阵列辅助插入突变(AIM)技术，并将该技术应用于全基因组发现和公共领域数据库开发，以在广泛的癌细胞系中调节药物反应的基因。在第一阶段的支持下，我们建议建立和验证一个通用的AIM基因发现平台，该平台将能够在低严格或高严格的条件下使用肯定或否定选择来明确地对癌症相关基因进行全基因组功能鉴定。目前还没有类似的平台，如果成功，这项工作将能够描述癌细胞中的药物反应机制，并将这一知识应用于转译研究项目。与我们来自克利夫兰诊所的生物顾问合作，我们将验证并应用AIM技术来揭示人类前列腺癌细胞中紫杉醇反应的决定因素。作为第一阶段计划的结果，AIM慢病毒载体和支持产品将作为现成的商业试剂发布，用于全基因组功能分析。 采用AIM技术的正向基因筛查有可能极大地简化基因功能的验证，并显著影响癌症疾病机制的分子解剖。AIM筛查在确定新的癌症药物靶点以进行治疗干预以及开发日益相关的药物发现范例方面具有相当大的希望。因此，我们预计这些工具箱将显著提高进行HT基因筛查的效率、经济性和简便性，并将为基础研究人员提供首选的、成本效益高的替代现有商业试剂的方法。

DESCRIPTION (provided by applicant): 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.