Gene expression associated with lentiviral expression of PRAME in normal hematopoietic progenitors.
ABSTRACT: To identify gene expression that distinguishes hematopoietic cells that express PRAME from those that do not, normal CD34+ cells with forced PRAME expression were compared to cells without PRAME expression in culture over time (days 4, 7, 14) using Affymetrix HU-133A microarrays Normal CD34+ progenitor cells not expressing PRAME were transduced with either PRAME cDNA (no 3' utr) or a corresponding empty vector control by lentiviral vector infection. Two replicates were performed for each condition (PRAME and control) at three experimental time points (days 4, 7, and 14 in culture). A single-stranded linear amplification protocol was used to process total RNA (one microgram) for the microarrays. This protocols is described in detail in PMID 14706461.
SUBMITTER: Janis L Abkowitz Katherine A GuthrieVivian G. OehlerTed GooleyMirjam T EppingJerald P RadichCarrie L CummingsVivian G OehlerDerek L StirewaltTaimei YangBrent L WoodPaula LadneEra Pogosova-AgadjanyanKathleen SaboYaping Shou
The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progres ...[more]
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Project description:Target genes of HOXA1 and HOXA9 were determined with the help of inducible HOX-ER constructs. For this purpose bone marrow was harvested from C57BL/6 mice and c-Kit positive progenitors were enriched by magnetic sorting (MACS technology, Miltenyi, Germany). After retroviral infection by spinoculation the cells were plated for three rounds in methylcellulose. Subsequently cells were maintained in liquid culture in RPMI1640 supplemented with murine recombinant cytokines at 100ng/ml (stem cell factor) and 10ng/ml (IL-3, IL-6, granulocyte/macrophage-colony stimulating factor) as well as 100nM 4-hydroxy-tamoxifen (TAM) for HOX activation.<br><br>For microarray analysis 4 independent cell lines were created for each HOX construct. RNA was isolated from cells in the presence of TAM and 72h after withdrawal of the inductor. Pairs of RNA replicates were pooled and two +TAM as well as two -TAM duplicates per HOX gene were hybridized to Agilent44K mouse expression arrays. Array processing and evaluation was done commercially according to Agilent specifications by ImaGenes (now SourceBioScience), Berlin, Germany.
Project description:Genome wide RNA-seq from pGM and HSCs in response to expression of the MLL-ENL fusion gene Examination of mRNA abundance in two cell types with or without induction of the MLL-ENL fusion gene (following 48h of culture)
Project description:Purpose: To characterize the genome-wide distribution of H3K79me2 in murine MN1 driven myeloid leukemia Methods: We performed Chip-seq for the H3K79me2 in leukemias isolated from moribund mice that had been injected with common myeloid progenitors (CMPs) transduced with MSCV-MN1-GFP Results: H3K79me2 is enriched at key loci that 1. are bound by MN1 in the data set of Heuser et al, (Cancer Cell. 2011 Jul 12;20(1):39-52.), 2. upregulated upon transduction with MN1, and lose expression upon deletion of the H3K79 methyltransferase Dot1l. Conclusions: A leukemogenic program in MN1 leukemias is marked by H3K79me2 and dependent on this mark ChIP-Seq for H3K79me2 using MN1 driven leukemias isolated from the bone marrow of moribund mice.