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

开发基于阵列的并行转录因子表达测定法

• 批准号: 7347355
• 负责人: Stephen Patrick Walton
• 金额: $ 18.09万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-06 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7347355
• 关键词: Abbreviations; Acquired Immunodeficiency Syndrome; Address; Affinity; Base Pairing; Behavior; Binding Proteins; Biological Assay; Biological Models; Cell Line; Cell physiology; Cells; Characteristics; Class; Complement; Computer Systems Development; Condition; Consensus; Consensus Sequence; Cyclic AMP; Cyclic AMP Response Element; Cyclic AMP-Responsive DNA-Binding Protein; DNA Binding; Detection; Development; Disease; Dissociation; EMSA; Electrophoretic Mobility Shift Assay; Elements; Environment; Equilibrium; Event; Fatty Acids; Future; Genetic; Genomics; Global Change; Hepatic; Hepatocyte; Human; In Vitro; Individual; Label; Lactate Dehydrogenase; Lactate Dehydrogenases; Lipopolysaccharides; Liver; Malignant Neoplasms; Measurement; Measures; Mediating; Mediator of activation protein; Methods; Modeling; Molecular; NF-kappa B; Non-Insulin-Dependent Diabetes Mellitus; Nonesterified Fatty Acids; Nuclear; Nucleic Acids; Peroxisome Proliferator-Activated Receptors; Phase; Polyacrylamide Gel Electrophoresis; Polymerase Chain Reaction; Predictive Value; Property; Protein Analysis; Proteins; RXR; Rate; Reading; Reproducibility; Research; Sampling; Sensitivity and Specificity; Signal Transduction; Solutions; Specificity; Standards of Weights and Measures; Sterols; Stimulus; System; TNF gene; Techniques; Testing; Time; Translating; Triglycerides; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Work; activating transcription factor 1; base; chicken DcoHalpha protein; concept; cytokine; cytotoxicity; extracellular; hepatoma cell; human TNF protein; improved; protein expression; receptor; response; tool; transcription factor

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.

描述（由申请人提供）：转录因子通常是信号级联的末端。因此，它们是细胞功能变化的最终介体，导致细胞环境变化。转录因子的不适当或不受管制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，对单元格的转录因子谱的完整理解将为该单元的当前功能以及将来可能发生的功能变化的强烈指示提供精心详细的描述。尽管这些信息及其预测价值很重要，但很少有工具提供敏感，可重复和并行的转录因子表达分析。建议解决这种巨大需求，利用基因组学的验证工具。我们提出的方法将将转录因子的溶液相检测与基于PCR的读数结合，以实现每个样品的数百个转录因子分子的检测限。当我们开始表征对肝细胞的细胞因子和脂肪酸施用后发生的信号事件（II型糖尿病模型），我们将使用该系统作为开发和改进分析技术的模型系统。拟议的工作的具体目的是i）量化TFS在MB-Cassettes中的识别序列的体外关联； ii）确定测量个体和多个TF的较低检测极限； iii）测量培养中HEPG2细胞刺激后TF水平变化。 转录因子是细胞呼吁对环境变化做出反应的细胞蛋白。转录因子表达改变通常是疾病状况的标志。我们提出的工作将为衡量转录因子表达的全球变化提供更好的方法，进而提供对疾病的更好理解和更好的治疗。