p53 target Genes in Apoptosis

p53 细胞凋亡的靶基因

• 批准号: 6718332
• 负责人: PENG LIANG
• 金额: $ 27.86万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6718332
• 关键词: DNA damage; RNA interference; apoptosis; computer simulation; differential display technique; gene expression; genetic mapping; green fluorescent proteins; mathematical model; microarray technology; neoplasm /cancer genetics; northern blottings; p53 gene /protein; polymerase chain reaction; serial analysis of gene expression; tumor suppressor genes

DESCRIPTION (provided by applicant): Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for a systematic gene expression analysis. Most of previous DD work has taken a "shot-gun" approach of identifying one gene at a time, with a limited PCR reactions set up manually, giving DD a low-tech and low-throughput image. With our newly solved DD mathematical model, which has been validated by computer simulations, global analysis of gene expression by DD technology is no longer a shot in the dark. After identifying the "rate-limiting" factors that contribute to the "noise" level of DD method, we have optimized the DD process with a new platform that incorporates fluorescent digital readout and automated liquid handling. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity and throughput in comprehensive and quantitative analysis of gene expression. We plan to apply this newly integrated FDD technology to conduct a systematic and comprehensive screening for p53 tumor-suppressor gene targets using two well-defined biological systems which features tetracycline regulated expression of wild-type p53 in both colon cancer and lung cancer cells that undergo rapid apoptosis upon p53 induction. The p53 target genes identified will be subjected to secondary screening processes, including the use of methods independent of FDD, and an additional cell system where endogenous p53 can be activated by DNA damaging agents. In the final phase of this study, three other technologies, namely, the inducible enhanced green fluorescence protein (EGFP) co-expression system, in-frame GFP fusion expression system and mammalian RNA interference (RNAi), will be incorporated to provide functional identification and sub-cellular localization of p53 target genes involved in apoptosis. We anticipate that this systematic and pioneering study will not only uncover many (if not all) additional target genes of the most important tumor-suppressor gene, but also will provide an experimental basis for an objective comparison of major technologies for analysis of differential gene expression, in terms of accuracy, comprehensiveness and throughput. Such a cross-platform comparison in studying the same biological system will be crucial in pinpointing the strength and weakness of each method and helpful for future improvement of the next generation technologies through complementation, integration and refinement.

描述（由申请人提供）： 差异显示（Differential Display，DD）是鉴定差异表达基因最常用的方法之一。尽管该方法对生物医学研究有很大的影响，但缺乏DD技术的自动化，以提高其系统基因表达分析的通量和准确性。以前的大多数DD工作都采取了一次识别一个基因的“猎枪”方法，手动设置有限的PCR反应，使DD技术含量低，通量低。通过我们新解决的DD数学模型，该模型已被计算机模拟验证，通过DD技术进行基因表达的全局分析不再是在黑暗中拍摄。在确定了有助于DD方法的“噪声”水平的“速率限制”因素后，我们已经用一个新的平台优化了DD过程，该平台结合了荧光数字读出和自动液体处理。由此产生的流线型荧光DD（FDD）技术在基因表达的全面和定量分析中提供了前所未有的准确性，灵敏度和通量。 我们计划应用这一新的集成FDD技术进行系统和全面的筛选p53肿瘤抑制基因的目标，使用两个定义明确的生物系统，其功能四环素调控表达的野生型p53在结肠癌和肺癌细胞，经历快速凋亡后p53诱导。鉴定的p53靶基因将进行二次筛选过程，包括使用不依赖于FDD的方法，以及另外的细胞系统，其中内源性p53可以被DNA损伤剂激活。在本研究的最后阶段，将结合其他三种技术，即诱导型增强型绿色荧光蛋白（EGFP）共表达系统、框内GFP融合表达系统和哺乳动物RNA干扰（RNAi），提供参与凋亡的p53靶基因的功能鉴定和亚细胞定位。我们预计，这项系统性和开创性的研究不仅将发现许多（如果不是全部）最重要的肿瘤抑制基因的其他靶基因，而且还将提供一个实验基础，客观比较主要技术的差异基因表达分析，在准确性，全面性和吞吐量。在研究同一生物系统时进行这种跨平台比较对于确定每种方法的优缺点至关重要，并有助于通过补充，整合和改进来改进下一代技术。