Sensitive and integrated microfluidic ChIP assays for studying transcriptional re

描述（由申请人提供）：转录失调经常参与癌症的发展。染色质免疫沉淀（ChIP）试验是用于检查体内转录因子-DNA相互作用的首选技术。目前的ChIP技术受到大量细胞的要求和大量人工处理导致的长测定时间的限制。这些问题实际上阻止了其用于从动物和患者提取的原代细胞。在这个项目中，我们将开发微流控ChIP检测方法，用于基于小鼠和人类的微量原始样本的研究。在本项目结束时，我们将实现约20-50个细胞的ChIP-qPCR和约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ChIP的灵敏度，我们将展示ChIP检测的两个创新应用：1。研究来自原发性肿瘤的细胞亚群，即肿瘤起始细胞（TIC）中的转录调控; 2.通过对单个活小鼠进行微创检查来监测转录调控的时间动态。这些实验无法使用当前的技术进行，并将产生独特的见解，提高在分子水平上对癌症发展的理解。

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.