Project description:Using RNA-seq technology, we quantitatively determined the expression profile of microRNAs during mouse terminal erythroid differentiation. CFU-E erythroid progenitors were isolated from E14.5 fetal liver as the Ter119, B220, Mac-1, CD3 and Gr-1 negative, C-Kit positive and 20% high CD71 population. Mature Ter119+ erythroblasts were isolated from E14.5 fetal liver as C-Kit negative and Ter119 positive population. Consistent with nuclear condensation and global gene expression shut down during terminal erythroid differentiation, we found that the majority of microRNAs are downregulated in more mature Ter119+ erythroblasts compared with CFU-E erythroid progenitors.
Project description:The S1 and S3 erythroid developmental subsets were isolated using flow cytometry and the cell surface markers CD71 and Ter119 as described by Pop et. al. 2010 (PMID: 20877475) Expression profiles for S1 and S3 subsets were generated using Affymetrix GeneChips. Results were used to identify genes that are differentially expressed during erythropoiesis. Single cell suspensions were prepared by mechanically dissociating whole fetal livers obtained from E12.5 to E13.5 Balb/C mouse embryos. Cells were stained for CD71, Ter119, and a cocktail containing lineage-specific antibodies. S1 and S3 erythroid developmental subsets were identified and isolated using flow cytometric sorting as described by Pop et. al. 2010 (PMID: 20877475). S1 and S3 subsets were isolated on 3 seperate days to generate total RNA (biological replicates). 20 ng of total RNA from each biological replicate was converted to cDNA, linearly amplified and biotinylated using Ovation reagents (Nugen, San Carlos, CA). Samples were hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). Microarray suite 5 (MAS5) processed sample data were normalized to the average of 18SRNA (AFFX-18SRNAMur/X00686_M_at), GAPDH (AFFX-GapdhMur/M32599_3_at) and M-NM-2-actin (1419734_at) expression values. These gene expression profiles were performed as part of the manuscript by Shearstone et. al. Global DNA Demethylation During Erythropoiesis In Vivo
Project description:Using RNA-seq technology, we quantitatively determined the expression profile of microRNAs during mouse terminal erythroid differentiation. CFU-E erythroid progenitors were isolated from E14.5 fetal liver as the Ter119, B220, Mac-1, CD3 and Gr-1 negative, C-Kit positive and 20% high CD71 population. Mature Ter119+ erythroblasts were isolated from E14.5 fetal liver as C-Kit negative and Ter119 positive population. Consistent with nuclear condensation and global gene expression shut down during terminal erythroid differentiation, we found that the majority of microRNAs are downregulated in more mature Ter119+ erythroblasts compared with CFU-E erythroid progenitors. Examination of microRNA expression profiles in 2 cell types
Project description:We used scRNAseq to profile CD71/CD24low fetal liver erythroid progenitor cells isolated by 2 distinct methods: FACS and immunomagnetic isolation. Cells from both isolation methods were hashtagged using Biolegend mouse hashtag antibodies and library prepped together on the 10X chromium platform with the 3'RNA v3 kit. We also performed CITE-seq to profile proteogenomic expression of CD117 and CD71 on lineage-depleted mouse fetal liver erythroid progenitor cells. CITE-seq was performed through a separate library prep on the 10X chromium platform with the 3'RNAv3 kit.
Project description:Analyses of gene expression by RNA-Seq in mouse E14.5 fetal liver burst-forming unit erythroid (BFU-E) cells untreated or treated by dexamethasone (DEX) with or without PPAR? agonist GW7647. RNA-Seq was performed on enriched populations of mouse BFU-E isolated from E14.5 fetal liver, as well as BFU-E enriched cells treated with Dex ± GW7647.
Project description:We over-expressed ESlncRNA (AK148461) in fetal liver erythroid progenitor cells (Lin-cells), followed by microarray analysis to examine the global changes of gene expression level. We showed that ESlncRNA has an anti-apoptotic activity during mouse erythropoiesis. Compare the gene expression level in vector transduced fetal liver erythroid progenitor cells (Lin-cells) with that in ESlncRNA transduced fetal liver erythroid progenitor cells (Lin-cells).
Project description:Exosc8 and Exosc9 are components of the exosome that establish a barricade to erythroid maturation. Here, we knocked down Exosc8 in fetal liver-derived erythroid progenitor cells to determine the cohort of Exosc8-regulated genes in erythroid cells. Freshly isolated fetal liver progenitor cells were infected with retrovirus expressing shRNA targeting either luciferase or Exosc8. Total RNA was isolated from these cells after 3 days ex-vivo culture, during which the cells underwent erythroid maturation.
Project description:Primary murine fetal liver cells were freshly isolated from day e14.5 livers and then sorted for successive differentiation stages by Ter119 and CD71 surface expression (ranging from double-negative CFU-Es to Ter-119 positive enucleated erythrocytes) [Zhang, et al. Blood. 2003 Dec 1; 102(12):3938-46]. RNA isolated from each freshly isolated, stage-sorted population was reverse-transcribed, labelled, and then hybridized onto 3' oligo Affymetrix arrays. Important erythroid specific genes as well as the proteins that regulate them were elucidated through this profiling based on coexpression and differential expression patterns as well as by extracting specific GO categories of genes (such as DNA-binding proteins). Abstract (submitted paper): rationale for expression profiling Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic developmentâbecause Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis while the single knockouts do not. These serine-threonine kinases phosphorylate, and consequently modify the functions of, several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferationâexplained in part by impaired cell cycle progression as well as increased apoptosisâand terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesisâsuch as alpha-globin and mitoferrin 1âdemonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.