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）测定是检测体内转录因子- dna相互作用的首选技术。目前的ChIP技术受到大量细胞的需求和大量人工处理导致的长时间分析的限制。这些问题实际上阻碍了它在从动物和病人身上提取的原代细胞上的应用。在这个项目中，我们将开发基于小鼠和人类微量初级样品的微流控芯片分析。在本项目结束时，我们将实现ChIP-qPCR的灵敏度为~20-50个细胞，ChIP-seq的灵敏度为~1000个细胞，用于使用原代细胞的转录因子结合研究。利用超高灵敏度的优势，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的转录调控，即肿瘤起始细胞（tic）；2. 监测时间动态转录调节通过微创检查一个活的老鼠。这些实验不能用目前的技术进行，将产生独特的见解，提高对分子水平上癌症发展的理解。

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.