Transcription indication reporter array technology

转录指示报告基因阵列技术

• 批准号: 6739251
• 负责人: XIN JIANG
• 金额: $ 17.44万
• 依托单位: PANOMICS, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6739251
• 关键词: biotechnology; genetic library; genetic regulatory element; genetic transcription; high throughput technology; microarray technology; nucleic acid hybridization; nucleic acid probes; nucleic acid purification; nucleic acid sequence; reporter genes; technology /technique development; transcription factor; transfection

DESCRIPTION (provided by applicant): Transcription factors (TFs) are cellular proteins responsible for controlling gene expression. Understanding the mechanism underlying the activation of these proteins will help us dissect cellular signaling pathways and determine the relationship between transcription activation and human diseases. Monitoring the activities of transcription factors in vivo usually relies on a genetic reporter construct that contains a cis-element corresponding to a specific transcription factor, a minimal promoter, and a reporter gene. When the transcription factor is activated, it binds to the cis-element and induces the expression of the reporter gene. Through enzymatic analysis of the reporter, the activity of the transcription factor can therefore be measured, and furthermore the level of transcriptional induction can be determined by comparison of control and treated cells. As the reporter is a single molecule, each assay can determine only one transcription factor's activity. Consequently, if multiple transcription factors need to be measured, the work will be tedious and time-consuming because it will involve multiple transfections and a repeat of the same assay over and over again. In the study, we propose to use a series of tag sequences as the reporter. We will create a library of reporter constructs, each containing a cis-element and a unique genetic tag sequence. The expression of reporter tags will be induced if their corresponding transcription factors are activated and bound to the cis-elements in host cells. The expressed tag sequences can then be determined with an array assay. With this technology, the simultaneous activation of multiple transcription factors in signal transduction pathways in vivo can be monitored at once.

描述（由申请人提供）： 转录因子（TF）是负责控制基因表达的细胞蛋白质。了解这些蛋白质激活的机制将有助于我们剖析细胞信号通路，并确定转录激活与人类疾病之间的关系。在体内监测转录因子的活性通常依赖于基因报告构建体，其含有对应于特定转录因子的顺式元件、最小启动子和报告基因。当转录因子被激活时，它与顺式元件结合并诱导报告基因的表达。通过对报道基因的酶分析，因此可以测量转录因子的活性，并且此外可以通过比较对照和处理的细胞来确定转录诱导的水平。由于报告分子是单个分子，因此每次测定只能确定一种转录因子的活性。因此，如果需要测量多个转录因子，则工作将是乏味和耗时的，因为它将涉及多次转染和一遍又一遍地重复相同的测定。在这项研究中，我们建议使用一系列的标签序列作为报告基因。我们将创建一个报告构建体库，每个构建体包含一个顺式元件和一个独特的遗传标签序列。如果相应的转录因子被激活并与宿主细胞中的顺式元件结合，则报告标签的表达将被诱导。然后可以用阵列测定法确定表达的标签序列。利用该技术，可以一次监测体内信号转导通路中多个转录因子的同时激活。