Grant: Collaborative Health Research Projects

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 amplificationof sequences in nanolitre volumes to generate sufficient material for standard sample indexing and automated library construction. Following sequencing to sufficient coverage, single cell miRNA expression levels will be determined by counting the number of unique barcodes for each gene, thereby allowing their absolute quantification independent of amplification biases. Epigenomic analyses of single cells will use on chip processing of histone DNA complexes and detection of H3K27me3 and H3K4me3 modifications at specific regulatory sites pre-identified by CHIP-SEQ. This will represent a truly interdisciplinary team effort offering a unique discovery-to-application experience for trainees at all levels as well as members of the biotech and clinical sectors who already recognize the commercialization and potential of the IP and products to begenerated