Project description:We report the genome wide distribution of H3K79 dimethylation in mouse MLL-AF6 positive leukemias to assess whether this epigenetic mark drives MLL-target gene expression. Examination of H3K79 dimethylation in bone marrow cells from sacrificed terminally ill MLL-AF6 positive leukemic mice. The retroviral MSCV-IRES-neo-MLL-AF6 construct was transduced into mouse bone marrow lineage negative Kit +, Sca + (LSK) cells and these cells were injected after G418 selection into irradiated syngenic mice to establish MLL-AF6 positive leukemias.
Project description:<p>We are studying the natural history, pathogenesis and treatment of patients with WHIM syndrome, an immunodeficiency disorder characterized by warts, hypogammaglobulinemia, recurrent infections and neutropenia usually due to autosomal dominant gain-of-function mutations in chemokine receptor <i>CXCR4</i>. We have identified a patient born with WHIM syndrome and the WHIM mutation <i>CXCR4<sup>R334X</sup></i> who has been disease-free for 20 years and who lacks <i>CXCR4<sup>R334X</sup></i> in myeloid cells, the cells that drive disease manifestations. She is a genetic and hematopoietic mosaic, since she still has the mutation in lymphoid cells and non-hematopoietic cells. Cytogenetics and microarray analysis revealed that the mechanism of loss of the mutation was deletion of the mutant allele from one copy of chromosome 2. Whole genome sequencing of patient neutrophil and skin fibroblast genomic DNA revealed that the mechanism of deletion was chromothripsis, a process of chromosome shattering resulting in deletions and rearrangements of the non-deleted chromosomal segments. In the patient, this process evidently occurred in a single hematopoietic stem cell (HSC), resulting in deletion of the disease allele <i>CXCR4<sup>R334X</sup></i> and one copy of 163 other genes on chromosome 2. This HSC evidently acquired a growth advantage and repopulated the HSC population and the myeloid lineage. Consistent with this, studies using gene targeted mice in competitive bone marrow transplantation experiments revealed that selective <i>Cxcr4</i> haploinsufficiency (inactivation of one copy of <i>Cxcr4</i> and not of any other genes) was sufficient to confer a strong engraftment advantage over bone marrow cells from wild type mice as well as bone marrow cells from a mouse model of WHIM syndrome. These results suggest that <i>CXCR4</i> knockdown may be a useful strategy to enhance bone marrow engraftment in the absence of toxic bone marrow conditioning regimens.</p>
Project description:Background: To define changes in gene expression from stem cells and early progenitor cells lacking histone deacetylase 3 (Hdac3), we purified bone marrow Lineage Negative, Sca1/cKit positive and Flt3 negative cells from wild type and Vav-Cre/Hdac3Flox/- mice. These lineage-specific knock out mice lack Hdac3 throughout the hematopoietic system. To ensure that only cells lacking Hdac3 were measured, we used a Lox-STOP-Lox-ROSA26-GFP transgene such that any cell containing active Cre also expresses GFP. Methods: Bone marrow cells were harvested from 10-30 mice and the lineage negative fraction was separated using the Lineage Cell Depletion Kit and MACS columns (Miltenyi Biotec). The lineage negative fraction was then stained with antibodies for flow cytometry and the GFP positive fraction of the LSK/Flt3 cells were sorted on a Becton Dickinson FACSAria. Total RNA was isolated from the sorted bone marrow cells using a PerfectPure RNA extraction kit (5 Prime). LSK/Flt3- cells pooled from 2 groups of 5 null mice were compared to LSK/Flt3- or LSK/Flt3+ cells pooled from 30 wild type mice. The expression of individual genes was verified using reverse transcriptase (RT) PCR. Conclusion: Hematopoietic stem and early progenitor cells fail to express gene that are typically turned on early during lymphoid development.
Project description:Global Expression profiling of non-committed lineage negative hematopoietic progenitors was performed on bone marrow aspirates from wild type control and mutant mice. Lineage negative (lin-) bone marrow progenitors were isolated from wild type or mutant Npm1cA/+, NrasG12D, Npm1cA/+;NrasG12D using MACS Microbeads (Miltenyl Biotec).
Project description:Global Expression profiling of non-committed lineage negative hematopoietic progenitors was performed on bone marrow aspirates from wild type control and mutant mice. Lineage negative (lin-) bone marrow progenitors were isolated from wild type or mutant Flt3ITD/+, Npm1cA/+;Flt3ITD/+ using MACS Microbeads (Miltenyl Biotec).
Project description:To investigate the effects of CaSR depletion or overexpression on gene expression in primary AML cells, we compared the gene expression profile of sorted lineage negative cells from the bone marrow of normal wildtype, CaSR knockout or CaSR overexpression mice.
Project description:Comparison of gene expression in the stromal cell compartment (Lineage negative, CD45 negative bone marrow cells) of C57B/L6 and B6.SJL (Ly5.1) mice. duplicate samples were analyzed per strain (two biological replicates) and each of the two samples was pooled from 5 mice
Project description:Bulk transcriptomes of granulocyte-macrophage progenitor cells isolated from bone marrow of Rnaseh2bfl/fl/ Vav-Cre and Cre-negative ctrl mice; The sorted bone marrow cells had the following immunophenotype: lineage- CD201 (EPCR)- CD117+ CD16/32+ CD34+
Project description:MLL-fusions may induce leukemogenic gene expression programs by recruiting the histone H3K79 methyltransferase to MLL-target promoters. We evaluated gene expression changes after cre-mediated loss of Dot1l in leukemia cells obtained from mice injected with MLL-9 transformed lineage negative bone marrow cells.