Project description:In BRE-GFP transgenic mice BMP activated cells are marked by GFP expression. Analysis of GFP+ and GFP- LSK-SLAM HSC enriched fractions from fetal liver and adult bone marrow shows the interinsic differences in the genetic program of these two HSC enriched fractions. RNAseq of GFP+ and GFP- LSK-SLAM cells from BRE-GFP transgenic mice Fetal liver and adult bone marrow
Project description:In BRE-GFP transgenic mice BMP activated cells are marked by GFP expression. Analysis of GFP+ and GFP- LSK-SLAM HSC enriched fractions from fetal liver and adult bone marrow shows the interinsic differences in the genetic program of these two HSC enriched fractions.
Project description:RNAs were isolated from FACS sorted ScxGFP positive cells and GFP negative cells of forelimbs at E13.5, and characterized by RNAseq
Project description:High levels of Hes1 expression are frequently found in BCR-ABL-positive chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC–like disease; however the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-kB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of Hes1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, these CMPs secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC–like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells. Common myeloid progenitors (CMPs; Lineage negative, c-Kit positive, Sca-1 negative, Fc-gamma-receptor low, CD34 positive fraction) were sorted with a FACSAria cell sorter (Becton Dickinson). Retroviruses were generated by transfecting Plat-E packaging cells with retrovirus vector pMYs-Hes1-IRES-GFP or empty vector (pMYs-IRES-GFP) using FuGENE 6 (Roche Diagnostics). Infection of retrovirus harboring Hes1 (pMYs-Hes1-IRES-GFP) or empty vector (pMYs-IRES-GFP) into progenitors was performed using RetroNectin (Takara Bio). Hes1-transfected CMPs and Mock-transduced CMPs were isolated 36 hours after infection with a FACSAria cell sorter. One sample of Hes1-transfected CMPs and one sample of mock-transduced CMPs were analyzed with GeneChip Mouse Genome 430 2.0 Array.
Project description:Comparison of Mpl-/- mouse LSK cells, either treated with control (GFP) or Mpl lentivirus. Lineage negative bone marrow cells were isolated and transduced and transplanted into Mpl-/- recipient mice. After transplantation and follow up mice were sacrificed and LSK (lineage negative, Sca-1 positive, cKit positive) cells were isolated by FACS. RNA was isolated using RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) and RNA was amplified for microarray hybridization using the Nugen Ovation system (Nugen Technologies, AC Bemmel, Netherlands). The resulting material was hybridized to Affymetrix Mouse 430 2.0 arrays. RMA normalization and summarization was performed in R 2.10 using Bioconductor packages. The aim was to show the normalization of Mpl associated gene expression. 3 control (GFP transduced) samples of Mpl -/- mouse LSK cells and 3 treatment (Mpl transduced) samples of Mpl -/- mouse LSK cells.
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:We used microarrays to profile global gene expression changes of Pou5f1-GFP-positive germ cells between E11.5 to E15.5. Germ cells were FACS-purified from gonadal single cell suspension based on Pou5f1-GFP expression. Three timepoints were included in this study: E11.5 (male/female), E13.5 (male) and E15.5 (male). For each timepoint, three biological replicates were analyzed. The Pou5f1-GFP-negative (non-germ cell) fraction of E13.5 (male) gonads was also included as a control.
Project description:Purpose: We characterized the role of EKLF in erythroblast island macrophages in E13.5 mouse fetal liver by analysing the transcriptomes of F4/80+ macrophages from WT and EKLF-/- by RNA-Seq. In addition we analyzed the transcriptomes of F4/80+ fetal liver macrophages from an EKLF/GFP mouse, where GFP acts as a surrogate for EKLF expression, to determine the genes enriched in EKLF/GFP+ macrophages compared to EKLF/GFP- macrophages where EKLF is not expressed. Finally, we used single cell RNA sequencing to resolve the heterogeneous population of F4/80+ macrophages from WT. Methods: For RNA-Seq from WT and EKLF-/- F4/80+ macrophages were FACS sorted from primary E13.5 mouse fetal livers and RNA was isolated using the Trizol method. F4/80+ macrophages were also FACS sorted from the EKLF/GFP mouse and the GFP+ and GFP- populations were collected. The GFP+ population had low cell numbers and therefore RNA was isolated using an Agilent RNA Nanoprep kit. For single cell sequencing, E13.5 fetal livers were stained with F4/80-PE antibody and the F4/80-PE+ cells were purified using an EZ-Sep PE selection kit (mouse). 25,000 cells were submitted for single cell sequencing for the Chromium V3 platform. Library preparation was done using the standard Illumina Truseq workflow, and libraries were sequenced in a Hiseq 2500 for WT and EKLF-/-, or Novaseq for EKLF/GFP and single cell sequencing. Results: We found that 1954 genes are differentially expressed in EKLF-/- F4/80+ macrophages vs WT and 2330 genes are differentially enriched in EKLF/GFP+ F4/80+ macrophages using DESeq2. Of these, 504 are common and constitute the EKLF-dependent gene expression program in F4/80+ fetal liver macrophages. We resolved the F4/80+ WT macrophages into 13 clusters based on gene expression and find that 23% of the total F4/80+ cells express EKLF. Conclusions: We find that the F4/80+ fetal liver macrophage are a unique cell type. We identify the EKLF-dependent gene expression program in these macrophages and determine an important transcription circuit governed by EKLF that constitute cell cycle and other Klf and Sp family transcription factors. Single cell sequencing showed a highly heterogeneous population of macrophages with activated macrophage as well as erythro-myeloid characteristics. Based on the expression of EKLF, we identified specific cell surface markers for EKLF+ F4/80+ macrophages.