p53 target Genes in Apoptosis

描述(由申请人提供)： 差异显示(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产生了巨大的影响，但对于系统的基因表达分析，DD技术一直缺乏自动化来提高其吞吐量和准确性。以前的大多数DD工作采取了一次识别一个基因的“枪炮”方法，通过手动设置有限的PCR反应，使DD获得了低技术和低通量的图像。有了我们新求解的DD数学模型，并经过计算机模拟验证，利用DD技术进行全球基因表达分析不再是白日做梦。在确定了导致DD方法“噪声”水平的“限速”因素后，我们通过一个结合了荧光数字读数和自动化液体处理的新平台优化了DD流程。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、灵敏度和吞吐量。 我们计划应用这一新集成的FDD技术，使用两个定义明确的生物系统对p53抑癌基因靶点进行系统和全面的筛选，这两个系统的特点是四环素调控野生型p53在结肠癌和肺癌细胞中的表达，这些细胞在p53诱导后经历快速凋亡。已确定的p53靶基因将经过二次筛选过程，包括使用独立于FDD的方法，以及一个额外的细胞系统，在该系统中，内源性p53可以被DNA损伤剂激活。在这项研究的最后阶段，其他三种技术，即可诱导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)将被用于提供参与细胞凋亡的P53靶基因的功能鉴定和亚细胞定位。我们预计，这一系统的开创性研究不仅将揭示最重要的抑癌基因的许多(如果不是全部)额外的靶基因，而且将为在准确性、全面性和吞吐量方面客观比较主要差异基因表达分析技术提供实验基础。在研究同一生物系统时，这种跨平台的比较将对找出每种方法的优缺点至关重要，并有助于未来通过互补、整合和完善来改进下一代技术。

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.