Development of a parallel, array-based transcription factor expression assay

描述（由申请人提供）：转录因子通常是信号级联的末端。因此，它们是由细胞环境变化引起的细胞功能变化的最终介质。转录因子的不适当或不受调节的表达与许多疾病有关，包括癌症、AIDS和II型糖尿病。因此，在任何时候对细胞的转录因子谱的完整理解将提供对该细胞当前功能的精细详细描述，以及对未来可能发生的功能变化的强烈指示。尽管这一信息及其预测价值的重要性，很少有工具存在，提供灵敏的，可重复的，和平行的转录因子表达分析。建议利用来自基因组学的经验证的工具来解决这一重大需求。我们提出的方法将结合联合收割机的解决方案相检测的转录因子与PCR为基础的读出，以实现检测限的数百个转录因子分子每个样品。由于我们已经开始表征向肝细胞（II型糖尿病模型）给予细胞因子和脂肪酸后发生的信号传导事件，因此我们将使用该系统作为开发和改进分析技术的模型系统。拟议工作的具体目标是：i）量化TF与MB盒中其识别序列的体外关联; ii）确定单个和多个TF测量的检测下限; iii）测量培养中HepG 2细胞刺激后TF水平的变化。 转录因子是细胞对环境变化做出反应的细胞蛋白质。改变的转录因子表达通常是疾病状况的标志。我们提出的工作将提供一种更好的手段来测量转录因子表达的全球变化，反过来，可以更好地理解和更好地治疗疾病。

DESCRIPTION (provided by applicant): Transcription factors are generally the termini of signaling cascades. As such, they are the ultimate mediators of change in cell function resulting from changes in the cellular environment. Inappropriate or unregulated expression of transcription factors has been implicated in many diseases, including cancer, AIDS, and Type II diabetes. As such, a complete understanding of the transcription factor profile of a cell at any time would provide an exquisitely detailed depiction of the current function of that cell as well as strong indications of functional changes that may occur in the future. Despite the importance of this information and its predictive value, few tools exist that provide sensitive, reproducible, and parallel transcription factor expression analysis. It is proposed to address this significant need, leveraging validated tools from genomics. Our proposed method will combine solution-phase detection of transcription factors with a PCR-based readout to achieve detection limits of hundreds of transcription factor molecules per sample. As we have begun to characterize the signaling events that occur upon administration of cytokines and fatty acids to hepatic cells, a model of Type II diabetes, we will use this system as a model system for development and refinement of the analytical technique. The specific aims of the proposed work are to i) quantify in vitro association of TFs to their recognition sequences in MB-cassettes; ii) determine the lower detection limits for measurements of individual and multiple TFs; iii) measure TF level changes following stimulation of HepG2 cells in culture. Transcription factors are cellular proteins that cells call upon to respond to changes in their environment. Altered transcription factor expression is often a hallmark of a disease condition. Our proposed work will provide a better means of measuring global changes in transcription factor expression and, in turn, allow for greater understanding of and better treatment of disease.