Sensitive and integrated microfluidic ChIP assays for studying transcriptional re

描述(申请人提供)：转录异常经常与癌症的发展有关。染色质免疫沉淀(ChIP)分析是检测体内转录因子-DNA相互作用的首选技术。目前的芯片技术存在细胞数量多、检测时间长、人工处理繁琐等问题。这些问题实际上阻碍了它在从动物和患者中提取的原代细胞上的使用。在这个项目中，我们将开发微流控芯片分析方法，用于基于来自老鼠和人类的少量初级样本的研究。到本项目结束时，我们将达到~20-50个细胞的CHIP-qPCR和~1000个细胞的CHIP-SEQ的灵敏度，用于使用原代细胞进行转录因子结合研究。利用超高灵敏度，我们将展示芯片分析的两个创新应用：1.研究原发肿瘤细胞亚群，即肿瘤起始细胞(TICS)的转录调控；2.通过对单个活体小鼠的微创检查，监测转录调控的时间动力学。这些实验不能使用目前的技术进行，并将产生独特的见解，在分子水平上提高对癌症发展的理解。

DESCRIPTION (provided by applicant): Dysregulated transcription is frequently involved in cancer development. Chromatin immunoprecipitation (ChIP) assay is the technique of choice for examining in vivo transcription factor-DNA interactions. The current ChIP technology is limited by the requirement of a large number of cells and the long assay time caused by extensive manual processing. These problems practically prevent its use on primary cells extracted from animals and patients. In this project, we will develop microfluidic ChIP assays for studies based on tiny amounts of primary samples from mice and humans. We will achieve a sensitivity of ~20-50 cells for ChIP-qPCR and ~1000 cells for ChIP-seq for transcription factor binding studies using primary cells by the end of this project. Taking advantage of the ultrahigh sensitivity, we will demonstrate two innovative applications of ChIP assays: 1. Study transcriptional regulation in a cellular subset from primary tumors, namely tumor-initiating cells (TICs); 2. Monitor temporal dynamics in transcriptional regulation by minimally-invasive examination of a single live mouse. These experiments cannot be conducted using current technology and will yield unique insights that improve the understanding of cancer development at the molecular level.