Lineage commitment and differentiation to a mature cell type are considered to be unidirectional and irreversible processes under physiological conditions. The commitment of haematopoietic progenitors to the B-cell lineage and their development to mature B lymphocytes critically depend on the transcription factor encoded by the paired box gene 5 (Pax5). Here we show that conditional Pax5 deletion in mice allowed mature B cells from peripheral lymphoid organs to dedifferentiate in vivo back to ea ...[more]
Project description:A common lymphoid progenitor cell culture system was established, maintaining a multipotent, proliferating state of CLPs. Cells can be cultured and expanded in vitro for several months, maintaining their in vitro and in vivo lymphoid differentiation potential. We were interested, whether cCLP are changing, with regards to their gene expression program, compared to in vivo CLPs, during their expansion in culture. Therefore we compared the gene expression profile of cultured cCLPs and their counterparts in vivo, by NextGenSeq. CLPs were sorted from bone marrow of 6-10 weeks old C57BL/6 mice (n=4). Cells were sorted for Lin-, IL7R+, Flk2+, CD27+, Ly6d- marker expressing CLPs by Flow Cytometry for gene expression analysis. In parallel the same cell population was sorted and cultured under cCLP culture conditions. After two months culture the cells sorted again for Lin-, CD27+, Ly6d- cells by Flow Cytometry for gene expression analysis (n=4). The analysis shows that cCLPs maintain a very similar transcription profile to ex vivo sorted common lymphoid progenitors. Overall design: Comparative gene expression of 2 cell types.
Project description:The transcription factor Pax5 is essential for B cell commitment in the mouse, where it represses lineage-inappropriate gene expression, while simultaneously activating the B cell gene expression program. We have performed a global gene expression screen of wild type and Pax5-deficient pro-B cells in an attempt to identify the crucial Pax5 targets in early B-lymphopoiesis. We have also included Rag1-/- and wild type (+/+) proB cells starved of the cytokine IL-7 for 4 hours as controls. Rag1-/- proB cells are incapable of further differentiation due to an absence of immunoglobulin recombination and IL-7 is the major cytokine regulating proB cell growth. Keywords: comparison of genetically modified cell-lines Overall design: Twelve arrays were used to compare a single Pax5+/+ proB cell line grown in the presence or abscence of IL-7 for 4 hours. 3 independently derived Pax5-/- proB cells lines and a single Rag1-/- pro B cell line were also analysed. cDNA were hybridised in a saturated loop design using dye swaps for each comparison, such that all samples were compared twice.
Project description:Human peripheral blood IFN-γ+IL-17+ (TH1/17) and IFN-γ–IL-17+ (TH17) CD4+ T cells display distinct transcriptional profiles in high-throughput transcription analyses. Compared to TH17 cells, TH1/17 cells have similar gene signatures to mouse pathogenic TH17 cells. Overall design: CD4+ T cells isolated from PBMC of healthy donors (n=5) were stimulated with PMA and ionomycin for 3hr. TH1/17, TH17, TH1 (IFN-γ–IL-17-) and DN (IFN-γ and IL-17 double negative) CD4+ T subsets were sorted utilizing a capture assay that separates live CD4+ T subsets based on differential secretion of IL-17 and/or IFN-γ. sorted CD4 T cell subsets were subjected to gene expression analysis using the nCounter Nanostring Custom Codeset HuTH17.
Project description:This array analysis is to study the regulation of target messages’ expression in in vitro cultured murine neutrophils versus miR-223 null neutrophils. Culture media was SILAC-IMDM for MS analysis. Overall design: Wild-type cultured neutrophils versus miR-223 null cultured neutrophils
Project description:To seek effects of inflammatory status and 5-aminosalicylic acid (5-ASA, mesalazine) exposure ex vivo on mRNA levels within rectal mucosal biopsies from patients with ulcerative colitis. A total of 12 biopsies were analysed, 3 biological replicates in each of 4 categories (inflamed with or without 5-ASA, non-inflamed with or without 5-ASA).