DNA methylation analysis of mouse embryonic fibroblasts (MEFs) and Thymidine treatment-induced MEFs (induced cells).
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE144272 | GEO | 2020/01/28
REPOSITORIES: GEO
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Project description:Investigation of differentially expressed genes in thymidine treatment-induced MEFs. Mouse embryonic fibroblasts (MEFs) were obtained at E14.5 embryos and supplied with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) with 15% FBS, 100 μg/ml streptomycin and 100 U/ml penicillin. Potential hematopoietic progenitor cells (CD45+CD41+CD31+c-Kit+) were negatively selected with Biotin labeled anti-CD45, anti-CD31, anti-CD41 and anti-c-Kit antibodies by magnetic beads. Hereafter, treated MEFs are referred to as 4neg MEFs for initiation of induction. 4neg MEFs were seeded on mitomycin (MMC)-treated MEFs as feeder cells and cultured in DMEM with 15% FBS, 100 μg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, 0.1 mM non-essential amino acids and 100 μM thymidine as induction medium for 6 days. Cells were then trypsinized and re-seeded to new feeder cells for another 6 days. Cells were positively selected with Biotin labeled anti-CD45 by magnetic beads followed with analysis.
2020-01-11 | GSE143418 | GEO
Project description:Whole livers were collected from mouse fetuses at embryonic day 13.5 (E13.5), and single-cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 20% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO) (generously provided by Kyowa Hakko Kirin Co. Ltd.). Megakaryocytes were harvested for RNA purification from a day-3 culture. CD41+ cells were enriched using biotinylated anti-CD41 antibody (Serotec; clone MWReg30) and streptavidin-coupled Dynabeads (Dynal Biotech ASA) from a culture of E13.5 fetal liver cells of WT and p45–/– mice. Total RNA was purified from the sorted CD41+ cells.
2013-08-07 | GSE35645 | GEO
Project description:Mouse embryonic fibroblasts (MEFs) were generated from 13.5-day-old embryos obtained from heterozygous PKBa mice intercrosses (Yang et al., 2003). Briefly, after dissection of head and visceral organs for genotyping, embryos were minced and trypsinized for 30 min at 37°C. Embryonic fibroblasts were then plated and maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal calf serum (FCS) (Life Technologies), 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in an atmosphere of 5% CO2. All experiments were performed with wild-type and PKBa-/- MEFs between 15-20 passages. To induce adipocyte differentiation, 2-day-postconfluent cells (day 0) were treated with DMEM supplemented with 10% FCS, 8 mg/ml biotin, 4 mg/ml pantothenate, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone and 10 mg/ml insulin (all from Sigma). Total RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer’s instructions. Keywords: repeat
2005-09-01 | GSE2746 | GEO
Project description:Heterogeneity among iPSC lines with regard to their gene expression profile and differentiation potential has been described and has been at least partly linked to the tissue of origin. We generated iPSCs from primitive (linneg) and non-adherent differentiated (linpos) bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and ESCs. In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles. However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction towards the hematopoietic lineage. All three BM-iPSC lines derived from undifferentiated cells yielded high proportions of cells expressing the hematopoietic differentiation marker CD41, and in two of these lines, high proportions of CD41+/CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in linpos BM-iPSC and FIB-iPSC lines. These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in two of the linneg BM-iPSCs only. Thus, in summary our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic redifferentiation for linneg BM-iPSC lines, thereby further supporting the notion of an epigenetic memory in iPSCs. Murine embryonic fibroblasts (MEFs) from C3H mice were cultured in low-glucose DMEM supplemented with 10% heat-inactivated fetal calf serum gold (PAA, Pasching, Austria), penicillin-streptomycin, 1 mM L-glutamine and 0.05 mM beta-mercaptoethanol on gelatine-coated dishes. C3H MEFs were grown to confluence, inactivated with 10 ug/ml Mitomycin C (Sigma) and used as feeder layers. Virus production was performed in a four plasmid-manner. Briefly, 3.5x10^6 293T cells were seeded 24h prior to transfection in 10 cm dishes. 293T cells were cultivated in high-glucose DMEM (Gibco) supplemented with 10% heat-inactivated FCS, penicillin-streptomycin and 1 mM L-glutamine. Cells were transfected with 5 ug lentiviral vector, 8 ug pcDNA3.GP.4xCTE (expressing HIV-1 gag/pol), 5 ug pRSV-Rev and 2 ug pMD.G (encoding the VSV glycoprotein) using the calcium phosphate method in the presence of HEPES and chloroquine. Supernatants were harvested 48h and 72h after transfection, filtered and subsequently 50x concentrated by ultracentrifugation. Titers determined based on real-time PCR, were in the range of 1-5x10^7/ml. For iPSC generation, bone marrow cells were isolated from femurs and tibias of Oct4-GFP transgenic mice (OG2) and immunomagnetically separated into lineage negative (Lin-) and lineage positive (Lin+) populations using the mouse lineage depletion kit (Miltenyi Biotec). Lin- cells were cultivated in serum-free StemSpan medium (Stem Cell Technology) supplemented with 2 mM L-glutamine, penicillin-streptomycin, 10 ng/ml mSCF, 20 ng/ml mTPO, 20 ng/ml, 20 ng/ml IGF-2 and 10 ng/ml FGF-1 (all Peprotech). Lin+ cells were cultivated in Iscove's modified eagle medium (IMDM), supplemented with 15% heat-inactivated FCS, 1 mM L-glutamine, penicillin-streptomycin, 100 ng/ml mSCF, 100 ng/ml mFLT3-L, 10 ng/ml hIL-3 and 100 ng/ml hIL-11. Both Lin- and Lin+ cells were pre-stimulated in the aforementioned media for 48 h. Thereafter, 2x10^5 Lin- and and Lin+ bone marrow cells were transduced on Retronection-coated plates (Takara) with lentiviral vectors encoding for human Oct4, Sox2, Klf4 and c-Myc using a multiplicity of infection (MOI) of 50 per virus. Twenty-four hours after transduction, media were supplemented with 2 mM valproic acid. Transduced bone marrow cells were kept in hematopoietic medium until 5 or 7 days post transduction (p.t.) and then transferred onto Mitomycin C-treated MEF feeders on gelatine-coated dishes. Henceforward, cells were cultivated in ES cell medium (knockout DMEM (Gibco), 15% ES-tested FCS, 1 mM L-glutamine, 0.1 mM non-essential amino acids (Gibco), 100 uM beta-mercaptoethanol (Sigma), penicillin-streptomycin and 103 units/ml leukemia inhibitory factor (LIF, provided by the Max-Planck-Institute, Munster, Germany). Upon appearance of GFP-positive ESC-like colonies, single colonies were picked based on morphology and GFP expression. Murine ESCs and iPSCs were cultured on Mitomycin C-treated MEF feeders in the aforementioned ES medium. Murine ESCs and iPSCs were passaged every 2-3 days. The murine embryonic fibroblast-derived iPSC lines (MEF-iPS, 3FLV2, 4FLV1) were generated by transduction of OG2-MEFs with the same lentiviral vector constructs using standard technology. For iPSC lines 3FLV2 and 4FLV1, complete reprogramming was demonstrated by alkaline phosphatase and SSEA1-staining, pluripotency factor expression and teratoma formation.
2012-08-10 | GSE29635 | GEO
Project description:Mouse embryonic fibroblasts (MEFs) were generated from 13.5-day-old embryos obtained from heterozygous PKBa mice intercrosses (Yang et al., 2003). Briefly, after dissection of head and visceral organs for genotyping, embryos were minced and trypsinized for 30 min at 37°C. Embryonic fibroblasts were then plated and maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% foetal calf serum (FCS) (Life Technologies), 100 units/ml of penicillin and 100 mg/ml of streptomycin at 37°C in an atmosphere of 5% CO2. All experiments were performed with wild-type and PKBa-/- MEFs between 15-20 passages. To induce adipocyte differentiation, 2-day-postconfluent cells (day 0) were treated with DMEM supplemented with 10% FCS, 8 mg/ml biotin, 4 mg/ml pantothenate, 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone and 10 mg/ml insulin (all from Sigma). Total RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer's instructions. Experiment Overall Design: Time course of DMEM treated WT and PKBa -/- MEFs in quadruplicate
2007-12-01 | E-GEOD-2746 | biostudies-arrayexpress
Project description:Whole livers were collected from mouse fetuses at embryonic day 13.5 (E13.5), and single-cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 20% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO) (generously provided by Kyowa Hakko Kirin Co. Ltd.). Megakaryocytes were harvested for RNA purification from a day-3 culture. CD41+ cells were enriched using biotinylated anti-CD41 antibody (Serotec; clone MWReg30) and streptavidin-coupled Dynabeads (Dynal Biotech ASA) from a culture of E13.5 fetal liver cells of WT and p45–/– mice. Total RNA was purified from the sorted CD41+ cells. Gene expression was measured in primary megakaryocytes cultured from p45-/- fetal livers and wild type fetal livers at E13.5. Three independent experiments were performed.
2013-08-07 | E-GEOD-35645 | biostudies-arrayexpress
Project description:In this study we combined previous transcriptomics and immunopeptidomics data with new proteomics data from untreated and IFNg-treated CRC PDOs to dissect mechanisms that lead to remodeling of the immunopeptidome through IFNg treatment (this is from the paper introduction, let me know if it needs any stylistic changes). · Experiment description: The cells were grown in DMEM/F12 media with 20% fetal bovine serum, 1X Glutamax, 100 units/ml penicillin/streptomycin and 2% matrigel, and treated with 600ng/mL IFNg for 48 hours before harvesting. Cells were washed twice with ice-cold PBS then snap-frozen.
2023-09-29 | PXD031634 | Pride
Project description:MCF7 and MDA-MB-231 breast cancer cell lines were cultured in DMEM-F12 containing 10% FBS (Lonza) 100U/ml penicillin and 100 µg/ml streptomycin (Lonza). Transfecting third generation packaging vectors using Poly-ethylenimine into HEK293T cells generated lentiviral particles (17). MCF7 and MDA-MB-231 cells were stably transduced with lentivirus containing pINDUCER20-FOXO3.A3, allowing doxycycline induced expression of constitutively active FOXO3 (FOXO3.A3). Cells were treated with 20% FBS or 10 µM PI3K inhibitor LY294002 (Selleckchem) for 16 hours to activate and inactivate the endogenous PI3K pathway, respectively. FOXO3.A3 expression was induced by 16 hours treatment with 10 ng/ml doxycycline.
2018-06-06 | GSE113479 | GEO
Project description:Whole fetal livers were collected from mouse fetuses at embryonic day 14.5 (E14.5), and single-cell suspensions were prepared by successive passage through 18-, 21 and 23-gauge needles. Fetal liver cells were maintained in Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO; Peprotech). After 5 days of culture, megakaryocytes were purified using a discontinuous bovine serum albumin gradient (BSA, Sigma–Aldrich; 3%, 1.5%, and 0%). Total RNA was isolated with Tri–Reagent (MRC) following manufacturer’s instructions, and its quality was assessed with ND–1000 Nanodrop (Peqlab) and on a 1.5% agarose gel.
2014-06-11 | GSE52141 | GEO
Project description:Purpose: The goals of this study are to use NGS to perform transcriptome profiling (RNA-seq) to find the difference of the transcriptomes of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts. Methods: MEFs were reprogramed into neurons by small chemical cocktails. Small chemical molecules were dissolved and diluted in DMSO and used at the following final concentrations: ISX9: 20 mM; Forskolin: 50 mM; CHIR99021: 20 mM; and I-BET151: 2 mM. In addition to the small molecules, the neuron induction medium (Neurobasal Medium) contains the following supplements: N2 (1X) and B27 (2X) supplements, GlutaMAX (1X), penicillin-streptomycin (100 µg/ml), bFGF (20 ng/ml), 100 µM cAMP, Non-essential Amino Acid (1X) and Trace element B (1X). MEFs were seeded to Matrigel-coated plate (1:30 dilution in pre-cold PBS and coat overnight at 4 oC, at a density of 200,000 cells per well in 6-well plate and 10,000 cells/well in 96 well plate. The MEFs were cultured in DMEM until confluent. When the cells are confluent, the DMEM was replaced with neuron induction medium with 4 small molecules. The induction medium was refreshed every two days for the first week and every 3 days for the remaining induction period until day 20. 3 biological replicates of total RNA were used for RNA-seq library preparation. Results: Using an optimized data analysis workflow, we identified neuronal gene programs induced during direct reprograming. The induction of neuronal programs were impaired in neurons from Actb-/- mouse embryonic fibroblasts. Conclusions: Our study shows that neuronal program induction was impaired in chemically induced neurons from Actb-/- mouse embryonic fibroblasts.
2018-04-27 | GSE113733 | GEO