Project description:Gene expression of LSK (lin-Sca-Kit+) hematopoietic stem cells from wild type mice was compared with LSK from Cebpa knock-in mutant mice (K/K, K/L, and L/L mutants). Fetal liver cells for each genotype were competitively transplanted into irradiant recipients. Donor-derived LSK cells were isolated by FACS sorting of recipient bone marrow. 3 biological replicates of each were generated and expression profiles were determined by hybridization to Affymetrix Moe430_2 arrays.
Project description:This experiment was performed to elucidate the effects of CDK6 on CALR-mutant Myeloproliferative Neoplasms. VavCre CALRdel52 knockin mice were crossed with full-body Cdk6-/- mice to obtain Cdk6-/- CALRdel52 mice. Bone marrow from mice of the four genotypes Cdk6+/+ CALR+/+ (wild-type), Cdk6+/+ CALRdel52 (CALR-mutant), Cdk6-/- CALR+/+ (Cdk6 knockout) and Cdk6-/- CALRdel52 (CALR-mutant with Cdk6 knockout) was isolated and stained for flow cytometry. To obtain megakaryocyte progenitors (MkPs) from the bone marrow, Lineage (CD11b, Gr-1, Ter119, CD3, CD19), c-Kit, sca-1, CD150 and CD41 surface markers were used. From the gating strategy: Lineage- c-Kit+ sca-1- CD150+ CD41+, MkPs were sorted into round-bottom collection tubes via fluorescence activated cell sorting (FACS). These cells were then used for RNA-isolation and subsequent RNA-Sequencing. The samples were harvested in two batches (Sample dates: \"February\" and \"April\").
Project description:Recurrent mutations in calreticulin (CALR) are present in 70-80% of JAK2 unmutated myeloproliferative neoplasms (MPN). Current models of CALR mutant MPNs are mainly based on cancer cell lines with ectopic overexpression or transgenic mouse models with a lack of data for primary human hematopoietic stem and progenitor cells (HSPCs) with endogenous CALR expression. Thus, we developed a CRISPR/Cas9 and AAV6-mediated knock-in approach to introduce the two most common CALR mutations (52 bp deletion, DEL; 5 bp insertion, INS) at the endogenous gene locus in human cord blood-derived HSPCs. We used these cells to investigate transcriptional changes induced upon CALR mutation acquisition in HSPCs in a prospective manner by performing RNA-sequencing 4 days after CRISPR-mediated knock-in.
Project description:Ezh1 is a protein member of PRC2. Ezh1 has been described as a functional repressor gene, such as its homologous Ezh2. We are investigating the role of Ezh1 in hematopoietic stem cells, aging, self-renewal and differentiation. We used microarrays to detail the global program of gene expression in LSK cells from mice with knocked-down expression of Ezh1. LSK cells from EZH1 knockout and control mice were marked and isolated by fluorescence-activated cell sorting. 2 replicates each.
Project description:Ezh1 is a protein member of PRC2. Ezh1 has been described as a functional repressor gene, such as its homologous Ezh2. We are investigating the role of Ezh1 in hematopoietic stem cells, aging, self-renewal and differentiation. We used microarrays to detail the global program of gene expression in LSK cells from mice with knocked-down expression of Ezh1.
Project description:We analyzed the effect of Calr deficiency on Mac1 and Gr1 positive bone marrow cells using hematopoietic cell specific Calr knockout (Mx-cre;Calrf/-) mice and control (Mx-cre;Calr+/+) mice.
Project description:Mutations in the endoplasmic reticulum (ER) chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We employed mass spectrometry proteomics to identify novel CALR-mutant interacting proteins. Mutant CALR caused mislocalization of binding partners and increased recruitment of FLI1, ERP57 and CALR to the MPL promoter to enhance transcription. CALR 52 mutant was also found to increase genome-wide recruitment of Fli1 to the chromatin. Overall, these results show that type 1 CALR mutant modulates Fli1 cellular localization and recruitment.
Project description:To understand kinase-dependent and kinase-independent functions of Cdk6 in the hematopoietic stem and progenitor compartment, mouse models carrying wild-type Cdk6 (Cdk6_WT), a homozygous knock-in of a kinase-inactivated variant of Cdk6 (Cdk6_K43M) or a homozygous Cdk6 knock-out (Cdk6_KO) were used. From these mice, lineage-negative, Sca1-positive, c-Kit-positive cells (LSK cells) were isolated by means of flow cytometry cell sorting. 30000 LSK cells per mouse were subjected to library prep for single cell RNA Sequencing using the Chromium NextGem Single Cell 5’ v2 Kit (10x Genomics, Pleasanton, CA, USA)