Array-Assisted Insertional Mutagenesis for Molecular Analysis of Cancer

描述（由申请人提供）：我们建议开发和验证一种技术，该技术将在低或高严格条件下使用正选择或负选择，对癌症相关基因进行明确的全基因组功能鉴定。这将把前向遗传学的研究范围扩展到一系列新的现象上。基因及其产物构成细胞表型的分子基础，其鉴定是癌症分子分析的根本问题。控制增殖、死亡、对治疗的反应、运动、与其他细胞和环境的相互作用的细胞因子已成为诊断和预后标志物，以及治疗干预的目标。正向遗传学是一种流行的方法，它使用遗传工具来揭示各种生物过程的调节剂。基于随机插入强调控启动子的插入诱变是一种多功能、经济高效、无偏倚和全面的体细胞正向遗传方法。然而，目前这种方法的实施只适用于严格的阳性选择条件，而不适用于更广泛的临床显著现象。此外，多个相关插入目标的大规模映射和验证仍然是整个方法的技术瓶颈。为了克服这些限制，我们将开发一种新的技术，将插入诱变的元素与微阵列分析的元素结合起来。微阵列组件将允许在高度复杂的细胞池中对插入物进行高通量映射。在不同的选择条件下，个体插入表达的积极或消极变化可以用来识别影响细胞行为的基因座。这些变化对插入启动子功能的依赖性可以用于大规模验证这些发现。我们将构建一系列合适的慢病毒载体，并将证实它们作为插入诱变剂的活性。我们将优化程序，以高通量格式监测单个突变等位基因的频率。最后，我们将应用新开发的工具和程序来发现基因，其产物决定前列腺癌对紫杉醇的反应，紫杉醇是唯一已知可以延长雄激素非依赖性前列腺癌患者生命的化疗化合物。我们将确定使细胞敏感或保护细胞不受药物影响的事件。这些发现的临床意义将在未来的研究中继续进行，同时我们的数据、工具和程序将与研究界共享。

DESCRIPTION (provided by applicant): We propose to develop and validate a technology that will 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. This will extend the reach of forward genetics to a principally new set of phenomena. Identification of the genes and their products, which form the molecular basis of cellular phenotypes, is the fundamental problem in molecular analysis of cancer. The cellular factors that control proliferation, death, response to therapy, motility, interaction with other cells and the environment have emerged as the diagnostic and prognostic markers, as well as the targets of therapeutic intervention. Forward genetics is a popular methodology that uses genetic tools to uncover the modulators of various biological processes. Insertional mutagenesis based on random insertion of a strong and regulated promoter is a versatile, cost-efficient, unbiased and comprehensive approach to forward genetics in somatic cells. However, the current implementations of this approach apply exclusively to the conditions of stringent positive selection, and are inapplicable to a wider range of clinically-significant phenomena. Moreover, large-scale mapping and validation of multiple relevant insertional targets remains the technical bottleneck of the whole approach. To overcome these limitations, we will develop a new technology, which combines the elements of insertional mutagenesis with those of microarray analysis. The microarray component will allow high-throughput mapping of the inserts in a highly complex pool of cells. Positive or negative changes in the representation of individual inserts could be used to identify the loci, which affect the cell behavior under various selective conditions. The dependence of the changes on the function of the inserted promoter could be used for large-scale validation of the findings. We will construct a series of appropriate lentiviral vectors and will confirm their activity as insertional mutagens. We will optimize the procedure to monitor the frequency of individual mutant alleles in a high-throughput format. Finally, we will apply the newly developed tools and procedures to the discovery of genes whose products determine the response of prostate carcinoma to Taxol, the only chemotherapeutic compound known to extend the life of patients with androgen-independent prostate cancer. We will identify the events that either sensitize or protect cells from the drug. The clinical significance of these findings will be pursued in future studies, while our data, tools and procedures will be shared with the research community.