Comparison of aged dormant and active hematopoietic stem cells
ABSTRACT: 4 months old Scl-tta; H2B-GFP mice where chased with Doxycyclin for 18 months from and then sacrificed at 22 months of age. GFP+ and GFP- cells, or dormant and active HSCs were then sorted and sequenced.
Project description:NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass, despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function. Messenger RNA was isolated from quadriceps muscle of mice from three different age groups and three different genotypes. Wildtype mice were aged 4, 7, and 24 months. Mice deficient for Nampt in skeletal muscle (mNKO) were aged 7 months. Mice overexpressing Nampt in skeletal muscle were aged 4 and 24 months.
Project description:Mouse embryonic stem cells can be maintained in a naive state of pluripotency by culture conditions containing inhibitors of Mek and Gsk3. Myc activity is essential for efficient cellular reprogramming. We genetically addressed the role of c-myc and N-myc in naive ESCs cultured in 2i by inducing the deletion of both genes using CRE-mediated recombination. Our findings show that myc activity controls the biosynthetic and proliferative machines of ESCs without significantly affecting the pluripotency network.
Project description:Somitogenesis is the segmentation of the developing embryonic body axis into somites and is guided by oscillating genes, which create waves of expression that travel across the presomitic mesoderm (PSM) from posterior to anterior. Upon arrival of a wave at the PSM's anterior end, a new somite is formed. To identify genes that are expressed in a wave-like pattern we dissected the PSM of four different mouse embryos (pre-turned), separated the left and right sides, and divided each into five segments, from posterior to anterior (sampling sites 1 to 5). Each segment was used to construct libraries for high-throughput RNA-sequencing. For one embryo, we also sequenced two somites.
Project description:We next sought to identify the transcriptional program that differentiates IL-9+Th2 cells from “conventional” Th2 cells. To this end, we selected representative Th1, Th17, Th2, and IL-9+Th2 clones (Figure 5A) and determined their transcriptome in the resting state and at different time points after activation using RNAseq. Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation. Human CD4+ T cells were isolated from PBMC by EasySep positive selection kit (Stemcell Technologies) according to manufacturer’s instruction. Positively selected CD4+ T cells were washed with PBS and stained for subsequent Th cell subset sorting. Memory Th cell subsets were sorted to over 90% purity according to their expression of chemokine receptors from CD45RA-CD25-CD8-CD3+ cells: Th1(CXCR3+CCR8-CCR6-CCR4-), Th2 (CXCR3-CCR8-CCR6-CCR4+), Th17 (CXCR3-CCR8-CCR6+CCR4+), Th9 (CXCR3-CCR8+CCR6-CCR4+). Single cell Th subset clones were directly sorted into 96well plate according to their expression of chemokine receptors (see above). Single cell clones were expanded and maintained by periodic restimulation with PHA (phytohaemaglutinine, 1 µg/ml, Sigma-Chemicals) and irradiated allogenic feeder cells (5x104/well) in culture medium. T cells were polyclonally activated using beads coated with antibodies against CD3, CD2, and CD28 (T cell/bead = 2:1, human T cell activation/expansion Kit, Miltenyi). Cell cultures were sampled before activation (time 0h) and 2, 4, 6, 9, 12, 24, and 48 hours after activation.
Project description:SW480 were stably transfected with an episomal plasmid expressing GFP and miRNA34 from a bidirectional doxycyclin regulatable promoter (Bornkamm et al Nucleic Acids Res. 2005 Sep 7;33(16)). Polyclonal cell lines were obtained by selection with Hygromycin at 350ug/ml for 10 days. The cell llnes identified as GFP only express GFP, whereas the cell lines identified as miRNA34a express both GFP and miRNA34 under doxycyclin control. For the present experiment, cells were treated with 1ug/ml Docycyclin for 72h. Cells were harvested and total RNA was isolated using Trizol (Invitrogen). After RNA cleanup (RNeasy, Qiagen) Affymetrix 133 Plus 2.0 micorarrays were hybridized using standard techniques. Keywords: Cell line transfection Overall design: 2 cell lines with transfected miRNA34a with doxycyclin treatment, 2 cell lines with miRNA34a but without doxycyclin treatment, 2 cell lines with transfected GFP with doxycyclin treatment, and 2 cell lines with GFP but without doxycylin treatment. One array for per sample. The log-transformed probe-set values and the results of the statistical analysis for each probe-set, and the associated README file, are included as Supplementary files.
Project description:RNA-Seq analysis of mouse cardiac transcriptome. RNA was isolated from the ventricles of 12-weeks-old male mice. Sequencing libraries were prepared using TrueSeq Stranded RNA LT Kit Ribo-Zero Gold (Illumina, 15032619). Libraries were pair-end sequenced on a MiSeq sequencer (Illumina) and mapped to the Mus musculus reference genome GRCm38 (https://support.illumina.com/sequencing/sequencing_software/igenome.html) using TopHat2 (Kim et al., 2013). Differential gene-expression analysis between controls and the EphB4 flox mice was performed using DESeq2 (Love et al., 2014). Read counts were obtained with HTSeq (Anders et al., 2015). Genes were considered as differentially expressed when the FDR-adjusted p-value was Ôëñ0.05.
Project description:SW480 were stably transfected with an episomal plasmid expressing GFP and miRNA34 from a bidirectional doxycyclin regulatable promoter (Bornkamm et al Nucleic Acids Res. 2005 Sep 7;33(16)). Polyclonal cell lines were obtained by selection with Hygromycin at 350ug/ml for 10 days. The cell llnes identified as GFP only express GFP, whereas the cell lines identified as miRNA34a express both GFP and miRNA34 under doxycyclin control. For the present experiment, cells were treated with 1ug/ml Docycyclin for 72h. Cells were harvested and total RNA was isolated using Trizol (Invitrogen). After RNA cleanup (RNeasy, Qiagen) Affymetrix 133 Plus 2.0 micorarrays were hybridized using standard techniques. Experiment Overall Design: 2 cell lines with transfected miRNA34a with doxycyclin treatment, 2 cell lines with miRNA34a but without doxycyclin treatment, 2 cell lines with transfected GFP with doxycyclin treatment, and 2 cell lines with GFP but without doxycylin treatment. One array for per sample. Experiment Overall Design: The log-transformed probe-set values and the results of the statistical analysis for each probe-set, and the associated README file, are included as Supplementary files.
Project description:Summary: Ribonuclease Inhibitor (RI also known as Rnh1) is a 50 kDa, ubiquitously expressed leucine-rich repeat (LRR) protein. It is localized in cytosol and binds to pancreatic-type ribonucleases and inhibit their function. However, the entire biological role for Rnh1 is unknown. We generated RNH1 knock out K562 cells by CRISPR/Cas9 method. Here we studied differential gene expression from wild type and RNH1 knock out K562 cells by RNA-Seq analysis. Overall design: Total RNA was isolated from wild type and RNH1 deficient K562 cells.
Project description:We report gene expression of Treg cells isolated from injured muscle in IL-33 vs PBS treated mice. Male Foxp3-GFP C57BL/6 reporter (2 months old) mice were injured intramuscularly with cardiotoxin/rIL-33 (0.3 ug/muscle). Tregs were sorted directly into Trizol from injured muscle 4 days post-injury. Gene expression profiling of muscle Tregs from IL-33 vs PBS injured mice.
Project description:Germline mutations in LKB1 predispose to hereditary Peutz-Jeghers Syndrome (PJS), manifesting with gastrointestinal polyposis. We discovered that conditional deletion of Lkb1 in stromal fibroblasts using Fsp1-Cre leads to expansion of stromal cells and gastrointestinal polyposis in mice. Here we have investigated gene expression signatures in the Fsp1-Cre;Lkb1fl/fl mouse polyps harbouring bi-allelic deletion of Lkb1 in stromal cells together with wild-type epithelium. We provide RNA-seq gene expression data of 6 polyps, 4 adjacent gastric mucosa samples and 5 wild-type gastric mucosa samples from littermate controls. Our experiment demonstrates e.g. activated cytokine signaling and inflammatory pathways in the polyps.