The development of single cell microfluidic technologies for analysis of microRNA expression and epigenetic state with application to hematopoietic stem cells

该项目将创造新的和强大的技术来评估基因组水平上单细胞的转录异质性。作为测试案例的原型例子是造血系统的干细胞，现在有令人信服的证据表明，不同的命运决定部分是由许多自我更新分裂的自我繁殖机制决定的。了解这些机制的分子基础是当前扩大体外造血干细胞、开发更安全、更有效的基因治疗方案和阐明个体患者白血病发生机制的基础。该技术将在高度纯化的正常小鼠和人类造血干细胞群体中进行开发、验证和原理验证实验，将使microRNA （miRNA）表达和表观基因组标记能够以高保真度和低成本在来自小样本的数百个单个细胞上进行检测。MiRNA分析将利用芯片上提取、条形码和PCR扩增

This project will create new and robust technology to assess the transcriptional heterogeneity of single cells at a genomic level. The prototype example to be used as a test case is the stem cell of the hematopoietic system where compelling evidence now indicates that different fate decisions are determined in part by mechanisms that are self-propagating through many self-renewal divisions. Understanding the molecular basis of these mechanisms is fundamental to current intense efforts to expand hematopoietic stem cells ex vivo, to develop safer and more effective gene therapy protocols, and to elucidate mechanisms of leukemogenesis in individual patients. The technology to be developed, validated and tested in proof-of-principle experiments on highly purified populations of normal mouse and human hematopoietic stem cells will enable microRNA (miRNA) expression and epigenomic marks to be examined on hundreds of individual cells from small samples with high fidelity and at low cost. MiRNA analyses will utilize on-chip extraction, barcoding and PCR amplification