Complete sequence-based pathway analysis by differential on-chip DNA and RNA extraction from a single cell

Abstract We demonstrate on-chip, differential DNA and RNA extraction from a single cell using a microfluidic chip and a two-stage lysis protocol. This method enables direct use of the whole extract, without additional washing steps, reducing sample loss. Using this method, the tumor driving pathway in individual cells from a colorectal cancer cell line was determined by applying a Bayesian computational pathway model to sequences obtained from the RNA fraction of a single cell and, the mutations driving the pathway were determined by analyzing sequences obtained from the DNA fraction of the same single cell. This combined functional and mutational pathway assessment of a single cell could be of significant value for dissecting cellular heterogeneity in tumors and analyzing single circulating tumor cells

Sequencing of human genomes extracted from single cancer cells isolated in a valveless microfluidic device

Sequencing the genomes of individual cells enables the direct determination of genetic heterogeneity amongst cells within a population. We have developed an injection-moulded valveless microfluidic device in which single cells from colorectal cancer derived cell lines (LS174T, LS180 and RKO) and fresh colorectal tumors have been individually trapped, their genomes extracted and prepared for sequencing using multiple displacement amplification (MDA). Ninety nine percent of the DNA sequences obtained mapped to a reference human genome, indicating that there was effectively no contamination of these samples from non-human sources. In addition, most of the reads are correctly paired, with a low percentage of singletons (0.17 ± 0.06%) and we obtain genome coverages approaching 90%. To achieve this high quality, our device design and process shows that amplification can be conducted in microliter volumes as long as the lysis is in sub-nanoliter volumes. Our data thus demonstrates that high quality whole genome sequencing of single cells can be achieved using a relatively simple, inexpensive and scalable device. Detection of genetic heterogeneity at the single cell level, as we have demonstrated for freshly obtained single cancer cells, could soon become available as a clinical tool to precisely match treatment with the properties of a patient's own tumor

The Ash1L methyltransferase is essential for normal hematopoiesis in the mouse

Histones have essential functions in the regulation of gene expression through epigenetic modifications of their N-terminal tails. Acetylation, methylation, phosphorylation, ubiquitination and other post-translational histone modifications constitute a complex spectrum of changes referred to as the "histone code" that promotes activation or induces repression of gene transcription. In Drosophila, Polycomb and Trithorax group proteins are histone methyltransferases that catalyze repressive (H3K27) and permissive (H3K4) histone 3 lysine methylation, respectively. In mammals, the Polycomb gene Bmi1 is essential for preventing premature lineage specification of hematopoietic stem cells and multipotent progenitors, and as such it is necessary to sustain the long-term self-renewal potential of blood-forming stem cells. The Mixed Lineage Leukemia (MLL) gene is the mammalian homologue of the Drosophila Trithorax (Trx) gene. MLL is required for the normal emergence of hematopoietic stem cells during embryogenesis and their homeostasis during adult life. In addition, MLL fusion proteins are involved in leukemic transformation. These examples illustrate the importance of identifying histone methyltransferases with critical non-redundant function in hematopoietic stem cells and other somatic stem cell populations. In this context, I discovered a novel essential hematopoietic function for Ash1L, the mammalian homologue of the fly Absent, small, or homeotic discs 1 (Ash1) gene. In the fly, Ash1 is a SET domain containing protein that was identified as a Trithorax family member and has been reported to have H3K4, H4K9, and H4K20 methyltransferase activity. In mammals, Ash1L’s biochemical effects are still debated, with studies suggesting H3K4 or H3K36 methyltransferase activity, both associated with transcriptional activation. To investigate the role of Ash1L in vivo, I studied mice with profoundly deficient Ash1L expression as a result of a "gene trap" strategy. These mice display various non-hematopoietic phenotypes including growth retardation and infertility. In the hematopoietic system, Ash1L deficiency resulted in the near complete exhaustion of the stem cell pool in young adult mice. While neonates displayed normal numbers of hematopoietic stem cells, lack of Ash1L impaired their normal repopulation capacity in bone marrow competitive transplantation experiments resulting in a profound tri-lineage hematopoietic defect. These findings indicate a critical cell-autonomous requirement for Ash1L in the maintenance of hematopoietic stem cells. To further understand the biological basis of Ash1L’s action in hematopoietic cells, it is essential to characterize its biochemical activity. For this purpose, I generated expression vectors that will allow me to purify the functional Ash1L domains and associated protein partners via immunoprecipitation or in-vivo biotinylation with the BirA enzyme. In addition, I generated retroviral vectors that will allow me to perform overexpression and rescue assays as a basis for Ash1L structure-function studies in hematopoietic tissues. By combining this approach with a detailed study of Ash1L-deficient hematopoietic stem cells, I anticipate that I will be able to understand the unique function of this protein in the hematopoietic system and in other mammalian tissue