Mx-Cre mediated deletion of SRF in murine hematopoietic LSK (Lin-Sca1+c-Kit+) cells
ABSTRACT: Analysis of hematopoietic LSK(Lin-Sca1+c-Kit+) cells lacking the Serum response factor (SRF) gene. Results provide insight into the role of SRF in regulating genetic programs important for hematopoietic stem cell development Overall design: Comparison of the gene expression profile between murine Lin-Sca1+c-Kit+ cells from Mx-Cre C57BL/6 Srf WT (3) and C57BL/6 Srf KO (3). Cells were sorted by flow cytometry and RNA was harvested and hybridized to Affymetrix MOE430A.
Project description:Analysis of hematopoietic LSK(Lin-Sca1+c-Kit+) cells lacking the Serum response factor (SRF) gene. Results provide insight into the role of SRF in regulating genetic programs important for hematopoietic stem cell development Comparison of the gene expression profile between murine Lin-Sca1+c-Kit+ cells from Mx-Cre C57BL/6 Srf WT (3) and C57BL/6 Srf KO (3). Cells were sorted by flow cytometry and RNA was harvested and hybridized to Affymetrix MOE430A.
Project description:To identify the molecular characterisitics of parallel lineage-biased MPP populations arising from hematopoietic stem cells (HSC) we conducted genome-wide analyses of hematopoietic stem, progenitor and mature myeloid cell populations using Affymetrix Gene ST1.0 arrays. Microarray analysis of 3-5 biological replicates of the indicated hematopoietic populations, isolated by FACS sorting from C57BL/6 mouse BM. Immunophenotypic definitions: Long-term HSC (HSCLT) (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150+); Short-term HSC (HSCST) (Lin-/cKit+/Sca1+/Flk2-/CD48-/CD150-); MPP2 (Lin-/cKit+/Sca1+/Flk2-/CD48+/CD150+); MPP3 (Lin-/cKit+/Sca1+/Flk2-/CD48+/CD150-); MPP4 (Lin-/cKit+/Sca1+/Flk2+); CMP (Lin-/cKit+/FcγR-/CD34+); GMP (Lin-/cKit+/FcγR+/CD34+); Pre-granulocyte (PreGr) (Mac1+/Gr1int); Granulocyte (Gr) (Mac1+/Gr1hi). HSC and GMP samples listed here were also used as controls for our related microarray study GSE48893.
Project description:The involvement of mature hematopoietic cells in disease pathogenesis is well recognized. However it is not clear how if and how primitive progenitors might contribute to inflammatory disease processes. This microarray experiment is used together with data from functional assays to determine how primitive progenitors are altered in a mouse model of autoimmune arthritis and how this in turn might contribute to the disease process. KSL cells were FACS sorted from 7 to 9 6-7 week old arthritic (KRNxG7) mice as well as from two strains of non-arthritic age-matched control mice: KRN and B6xG7 mice. Cells were sorted using identical conditions and identical sorting gates. To verify the primitive status of the KSL cells, Lin+ cells were also MACS sorted from these same mice. All the mice used in this study were C57BL/6 background strain. G7 mice are congenic with C57BL/6 but with MHC II I-Ab replaced with MHC II I-Ag7.
Project description:Runx/Cbfb heterodimers play important roles in the development of hematopoietic cells in mouse embryos and adults. In order to identify genes that are regulated by Runx/Cbfb, we purified Lin– c-kit+ Sca1+ (LSK) cells and Lin– c-kit+ Sca1– CD16/32+ (GMP) cells from Vav1-iCre x Cbfb(F/F) and Vav1-iCre x Cbfb(F/+) mice and profiled gene expression using microarray. 200,000 LSK and GMP cells were purified separately from two 7 week old Vav1-iCre x Cbfb(F/F) mice and two Vav1-iCre x Cbfb(F/+) mice by cell sorting. The purity was higher than 98%. Total RNA was extracted using the NucleoSpin RNA XS kit (Macherey-Nagel), amplified using a PicoSL RNA amplification kit (Nugen) and biotinylated with Encore biotin module (Nugen). Labeled RNA was hybridized to Mouse Gene 1.0ST microarrays (Affymetrix) according to the manufacturer’s instruction.
Project description:The Ikaros zink finger transcription factor is a critical regulator of the hematopietic system, and plays an important role in the regulation of the development and function of several blood cell lineages. We used microarrays to characterize how Ikaros deficieny affects global transcription in the hematopoietic stem and progenitor cells from wild-type and Ikaros mutant mice. lin- Sca1+ c-Kit+ (LSK) cells, that contains the hematopoietic stem cells and the multipotent progenitor cells, were sorted by FACS from the bone marrow of 6-7 weeks old WT and IkL/L mutant mice. Total RNA extracted from these cells was subjected to transcriptome analysis.
Project description:To compare the impact of hematopoietic-specific Brpf1 gene inactivation, LSK (Lin-Sca1+cKit1+) cells were sorted from wild-type and Brpf1-null fetal liver cells for RNA-Seq. Four E14.5 embryos were used to pool sufficient LSK cells for total RNA isolation and subsequent sequencing on HiSq2500. Two independent pairs of wild-type and mutant RNA samples (each of which contained LSK cells pooled from four embryos) were used for oligo-dT primed RNA Seq.
Project description:Hematopoietic stem cells (HSC) are heterogeneous in their ability to durably reconstitute the blood system. Epigenome surveys along the hematopoietic hierarchy and within the HSC compartment have revealed distinct epigenomic states associated with functionally defined hematopoietic cell types. However, the degree to which epigenomic states vary within functionally defined hematopoietic populations is still largely unknown. To address this question we performed single-cell genome-wide DNA methylation profiling of linage negative Sca+cKit+ (LSK) and EPCR+CD45+CD48-CD150+ (ESLAM) hematopoietic stem cell populations purified from adult mouse bone marrow. For this we developed a bisulfite based whole genome protocol suitable for use on single index sorted mammalian cells. Analysis of the resulting data from 148 single cells revealed that the methylomes of single HSCs show characteristics consistent with those defined from bulk populations. Comparing methylation states across single HSCs revealed a significant reduction in CpG state adjacency compared to bulk estimates and that on average 13% of CpGs exist in the opposite methylation state between any two cells within both surface marker defined HSC containing populations. Utilizing a novel analytical approach designed to address missing data inherent in single cell assays we identified an epigenetically distinct subpopulation of cells present in both the LSK and ESLAM populations. Enrichment analysis of genes associated with this distinct epigenetic state revealed pathways consistent with hematopoietic stem cell function, and integration with single-cell RNA-seq data defined putative surface proteins that mark this population, including the previously identified hematopoietic marker, NRXN1. Overall design: One sample, no controls
Project description:The transcription factor Evi1 is essential for the formation and maintenance of hematopoietic stem cells, and induces clonal dominance with malignant progression upon constitutive activation by chromosomal rearrangements or transgene integration events. To understand the immediate and adaptive response of primary murine hematopoietic cells to the transcriptional upregulation of Evi1, we developed an inducible lentiviral vector system with a robust expression switch. We found that Evi1 delays differentiation and promotes survival in myeloid culture conditions, orchestrating a battery of genes involved in stemness (Aldh1a1, Ly6a [Sca1], Abca1, Epcam, among others). Importantly, Evi1 suppresses Cyclins and Cyclin-dependent kinases (Cdk), while it upregulates Cdk inhibitors, inducing quiescence in various proliferation-inducing cytokine conditions and operating in a strictly dose-dependent manner. Hematopoietic cells with persisting Evi1-induction tend to adopt a relatively low expression level. We thus classify Evi1 as a dormancy-inducing oncogene, likely requiring epigenetic and genetic compensation for cell expansion and malignant progression. Lin- Rosa26rtTA cells were isolated, transduced in S3F11 cytokines, induced the next day with DOX [1 μg/ml] and 20 hours later sorted for negative/low or highly EGFP expressing cells, from which total RNA was extraced and subjected to Microarray Analysis
Project description:Chemokine signaling is important for the seeding of different sites by hematopoietic stem cells during development. Serum Response Factor (SRF) controls multiple genes governing adhesion and migration, mainly by recruiting members of the Myocardin-Related Transcription Factor (MRTF) family of G-actin regulated cofactors. We used vav-iCre to inactivate MRTF-SRF signaling early during hematopoietic development. In both Srf- and Mrtf-deleted animals, hematopoiesis in fetal liver and spleen is intact, but does not become established in fetal bone marrow. Srf-null HSC/Ps (hematopoietic stem/progenitor cells) fail to effectively engraft in transplantation experiments, exhibiting normal proximal signaling responses to SDF-1, but reduced adhesiveness, F-actin assembly, and reduced motility. Srf-null HSC/Ps fail to polarise in response to SDF-1, and cannot migrate through restrictive membrane pores to SDF-1 or Scf in vitro. Mrtf-null HSC/Ps were also defective in chemotactic responses to SDF-1. MRTF-SRF signaling is thus critical for the response to chemokine signaling during hematopoietic development. Strand specific RNA sequencing (RNA-seq) in sorted WT and SRF deleted LSK cells with or without a 30 minute SDF stimulation and validation by qRT-PCR