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，它们是细胞环境变化导致细胞功能变化的最终介质。转录因子不适当或不受调控的表达与许多疾病有关，包括癌症、艾滋病和II型糖尿病。因此，在任何时候，对细胞转录因子谱的全面了解将提供对该细胞当前功能的精确详细描述，以及对未来可能发生的功能变化的强烈指示。尽管这些信息及其预测价值很重要，但很少有工具可以提供敏感的、可重复的、平行的转录因子表达分析。建议利用基因组学的验证工具来解决这一重大需求。我们提出的方法将结合转录因子的液相检测与基于pcr的读数，以达到每个样品数百个转录因子分子的检测极限。由于我们已经开始描述细胞因子和脂肪酸给肝细胞时发生的信号事件，这是II型糖尿病的一个模型，我们将使用该系统作为开发和改进分析技术的模型系统。所提议的工作的具体目的是：i)量化tf与其在mb磁带中的识别序列的体外关联；ii)确定单个和多个TFs测量的下限；iii)测定培养HepG2细胞刺激后TF水平的变化。

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.