Array-Assisted Insertional Mutagenesis for Molecular Analysis of Cancer

用于癌症分子分析的阵列辅助插入突变

• 批准号: 7586366
• 负责人: Eugene S Kandel
• 金额: $ 27.17万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7586366
• 关键词: Address; Affect; Alleles; Biological Models; Biological Process; Cancerous; Cell model; Cells; Cessation of life; Clinical; Communities; Complex; Data; Dependence; Detection; Development; Diagnostic; Dose; Drug resistance; Elements; Engineering; Ensure; Environment; Event; Feline Immunodeficiency Virus; Frequencies; Future; Generations; Genes; Genetic; Genetic Determinism; Genetic Transcription; Genome; Goals; Human; Human Genome; Hybrids; In Vitro; Individual; Insertional Mutagenesis; Investments; Lentivirus Vector; Libraries; Life; Ligation; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Maps; Mediating; Methodology; Microarray Analysis; Molecular; Molecular Analysis; Monitor; Mutagenesis; Mutagens; Mutate; PC3 cell line; Paclitaxel; Pathway interactions; Patients; Performance; Pharmaceutical Preparations; Phenotype; Play; Procedures; Process; Prognostic Marker; Prostate carcinoma; Protocols documentation; Reagent; Research; Resistance; Role; Series; Somatic Cell; Subfamily lentivirinae; Techniques; Technology; Testing; Therapeutic; Therapeutic Intervention; Transcript; Validation; Vertebral column; Work; androgen independent prostate cancer; base; biological systems; cancer cell; cell behavior; cell growth; cell motility; cellular transduction; clinically significant; cost; cost effective; fitness; gene discovery; genetic technology; genome wide association study; genome-wide; high throughput technology; improved; mutant; neoplastic; new technology; next generation; novel therapeutics; prognostic; promoter; response; technology development; tool; trait; tumor; vector

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.

描述（由申请人提供）：我们建议开发和验证一种技术，该技术将能够在低或高严格性条件下使用阳性或阴性选择对癌症相关基因进行明确的全基因组功能鉴定。这将把正向遗传学的研究范围扩展到一系列新的现象。构成细胞表型分子基础的基因及其产物的鉴定是癌症分子分析中的基本问题。控制增殖、死亡、对治疗的反应、运动性、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靶点。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体细胞正向遗传学中通用、经济、无偏倚和全面的方法。然而，这种方法的当前实现仅适用于严格的阳性选择的条件，并且不适用于更广泛的临床显著现象。此外，多个相关插入靶标的大规模定位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，它结合了插入突变的元素与微阵列分析。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。单个插入片段的表达的正或负变化可用于鉴定在各种选择条件下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，并将确认其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其产物决定前列腺癌对紫杉醇的反应的基因，紫杉醇是已知的唯一一种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免受药物影响的事件。这些研究结果的临床意义将在未来的研究中进行，而我们的数据，工具和程序将与研究界共享。