Next Generation Sequencing Analysis of chemically reprogrammed osteoblasts from Beta-actin Wildtype (WT) Heterozygotes (HET) and knockout (KO) Mouse Embryonic Fibroblast (MEFs)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE135392 | GEO | 2019/08/06
REPOSITORIES: GEO
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Project description:Next Generation Sequencing Analysis of chemically reprogrammed osteoblasts from Beta-actin Wildtype (WT) Heterozygotes (HET) and knockout (KO) Mouse Embryonic Fibroblast (MEFs)
| PRJNA558732 | ENA
Project description:In the filamentous fungus Podospora anserina, cell death by incompatibility can be monitored using the conditional self-incompatible (SI) het-R het-V strain. SI strains are homokaryotic strains bearing incompatible het genes in all nuclei. The co-expression of these het genes triggers cell death in all the cells and hence in the entire mycelium. The het-R het-V SI strain, bearing the two incompatible het-R and het-V genes, proved particularly convenient as cell death triggering is thermosensitive (J. Labarère et al., 1973, C R Acad Sci Hebd Seances Acad Sci D, 276) and is induced by a simple shift in growth temperature (from 32°C to 26°C).The development of this cell death reaction has been recorded for 4 h after transfer to the restrictive temperature. A time course (T0h to T4h) of the transcriptomic response to the temperature shift has been explored in parallel within SI (het-R het-V) and wild type (compatible het-r hetV) s strains.
2013-01-01 | GSE21659 | GEO
Project description:In the filamentous fungus Podospora anserina, cell death by incompatibility can be monitored using the conditional self-incompatible (SI) het-R het-V strain. SI strains are homokaryotic strains bearing incompatible het genes in all nuclei. The co-expression of these het genes triggers cell death in all the cells and hence in the entire mycelium. The het-R het-V SI strain, bearing the two incompatible het-R and het-V genes, proved particularly convenient as cell death triggering is thermosensitive (J. LabarM-CM-(re et al., 1973, C R Acad Sci Hebd Seances Acad Sci D, 276) and is induced by a simple shift in growth temperature (from 32M-BM-0C to 26M-BM-0C).The development of this cell death reaction has been recorded for 4 h after transfer to the restrictive temperature. A time course (T0h to T4h) of the transcriptomic response to the temperature shift has been explored in parallel within SI (het-R het-V) and wild type (compatible het-r hetV) s strains. With Platform GPL10116 : reference design with 2 strains: a WT strain and het-R het-V self-incompatible strain (J. LabarM-CM-(re et al., 1973, C R Acad Sci Hebd Seances Acad Sci D, 276). Each strain with four biological replicates; the common reference is a pool of four conditions M48h, M96h, C48h and C96h ; Conditions are labelled in Cy3 and the common reference in Cy5.
2013-01-01 | E-GEOD-21659 | biostudies-arrayexpress
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
Project description:Transcriptomic profile of germinal center B cells from conditional GC-specific (Cg1-Cre) Crebbp-HET, Kmt2d-HET, and compound Crebbp-HET/Kmt2d-HET mice, with littermate controls
| PRJNA909299 | ENA
Project description:We identified four transcriptional factors, Runx2, Osx, Dlx5, and ATF4, that rapidly and efficiently reprogram mouse fibroblasts derived from 2.3 kb type I collagen promoter-driven green fluorescent protein (Col2.3GFP) transgenic mice into induced osteoblast cells (iOBs). The global transcriptome profiling validated that iOBs resemble primary osteoblasts. Genome-wide DNA methylation analysis demonstrates that within differentially methylated loci, the methylation status of iOBs more closely resembles primary osteoblasts than mouse fibroblasts. We further demonstrate that Col2.3GFP+ iOBs have transcriptome profiles similar to GFP+ cells derived from Col2.3GFP transgenic mouse bone chips.
2020-01-01 | GSE139907 | GEO
Project description:Safety issues associated with transcription factors or viruses may be avoided with the use of chemically induced pluripotent stem cells (CiPSCs), thus promoting their clinical application. Previously, we had successfully developed and standardized an induction method using small-molecule compound, with simple operation, uniform induction conditions, and clear constituents. In order to verify that the CiPSCs were indeed reprogrammed from mouse embryonic fibroblasts (MEFs), and further explore the underlying mechanisms, FSP-tdTomato mice were used to construct a fluorescent protein-tracking system of MEFs, for revealing the process of CiPSC reprogramming. CiPSCs were identified by morphological analysis, mRNA, and protein expression of pluripotency genes, as well as teratoma formation experiments. Results showed that after 40-day treatment of tdTomato-MEFs with small-molecule compounds, the cells were presented with prominent nucleoli, high core-to-cytoplasmic ratio, round shape, group and mass arrangement, and high expression of pluripotency gene. These cells could differentiate into three germ layer tissues in vivo. As indicated by the above results, tdTomato-MEFs could be reprogrammed into CiPSCs, a lineage that possesses pluripotency similar to mouse embryonic stem cells (mESCs), with the use of small-molecule compounds. The establishment of CiPSC lineage, tracked by fluorescent protein, would benefit further studies exploring its underlying mechanisms. With continuous expression of fluorescent proteins during cellular differentiation, this cell lineage could be used for tracking CiPSC transplantation and differentiation into functional cells.
| S-EPMC6323470 | biostudies-literature
Project description:Osteoarthritis (OA) is the most common joint disease and this is a major cause of joint pain and disability in the aging population. Its etiology is multifactorial (i.e., age, obesity, joint injury, genetic predisposition), and the pathophysiologic process affects the entirety of the joint (Martel-Pelletier J et al. Osteoarthritis. Nature reviews Disease primers. 2016;2:16072). Although it is not yet clear if it precedes or occurs subsequently to cartilage damage, subchondral bone sclerosis is an important feature in OA pathophysiology (Goldring SR et al. Changes in the osteochondral unit during osteoarthritis: structure, function and cartilage-bone crosstalk. Nat Rev Rheumatol. 2016;12:632-44). It is characterized by local bone resorption and the accumulation of weakly mineralized osteoid substance (Bailey AJ et al. Phenotypic expression of osteoblast collagen in osteoarthritic bone: production of type I homotrimer. Int J Biochem Cell Biol. 2002;34:176-82). Subchondral bone sclerosis is suspected to be linked to cartilage degradation, not only by modifying the mechanical stresses transmitted to the cartilage, but also by releasing biochemical factors with an activity on cartilage metabolism (Sanchez C et al. Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts. Osteoarthritis Cartilage. 2005;13:979-87; Sanchez C et al. Subchondral bone osteoblasts induce phenotypic changes in human osteoarthritic chondrocytes. Osteoarthritis Cartilage. 2005;13:988-97; Westacott CI et al J. Alteration of cartilage metabolism by cells from osteoarthritic bone. Arthritis Rheum. 1997;40:1282-91. We have previously demonstrated that osteoblasts isolated from subchondral OA bone exhibited an altered phenotype. More precisely, we showed that osteoblasts coming from the thickening (called sclerotic, SC) of subchondral bone located just below a cartilage lesion produced higher levels of alkaline phosphatase, interleukin (IL)-6, IL-8, prostaglandinE2, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-9 and transforming growth factor(TGF)-β1 and type I collagen than osteoblasts coming from the non-thickening neighboring area (called non-sclerotic area, NSC) (Sanchez C et al. Phenotypic characterization of osteoblasts from the sclerotic zones of osteoarthritic subchondral bone. Arthritis Rheum. 2008;58:442-55; Sanchez C et al. Regulation of subchondral bone osteoblast metabolism by cyclic compression. Arthritis Rheum. 2012;64:1193-203.) To compare secretome of cells living in different in vivo conditions is useful, not only to better understand the pathological mechanisms underlying changes in OA subchondral bone, but also to identify soluble biomarkers potentially reflecting these changes. Using our well-characterised human subchondral osteoblast culture model, we compared the secretome of osteoblasts coming from sclerotic and non sclerotic OA subchondral bone. This approach allowed to identify changes in secretome that contribute to explain some subchondral bone abnormalities in OA and to propose osteomodulin and fibulin-3 as potential biomarkers of OA subchondral bone remodelling.
2018-03-20 | PXD008494 | Pride
Project description:Whole hearts from wild-type and Na,K-ATPase alpha 1 het. mice. Adult male, 8-16 weeks old on a 129/BSwiss background. Keywords: repeat sample
2004-11-19 | GSE1134 | GEO
Project description:PIWI-interacting (pi) RNAs provide silencing of transposable elements (TE) in the germline. Drosophila telomeres are maintained by transpositions of specialized telomeric retroelements. piRNAs generated from sense and antisense transcripts of telomeric elements provide telomere length control in the germline. Previously, we have found that antisense transcription of the major telomeric retroelement HeT-A is initiated upstream of the HeT-A sense transcription start site. Here, we performed a deletion analysis of the HeT-A promoter and show that common regulatory elements are shared by sense and antisense promoters of HeT-A. Therefore, the HeT-A promoter is a bidirectional promoter capable of processive sense and antisense transcription. Ovarian small RNA data shows that a solo HeT-A promoter within an euchromatic transgene initiates divergent transcription of transgenic reporter genes and subsequent processing of these transcripts into piRNAs. These events lead to the formation of a divergent uni-strand piRNA cluster at solo HeT-A promoters, in contrast to endogenous telomeres that represent strong dual-strand piRNA clusters. Solo HeT-A promoters are not immunoprecipitated with Heterochromatin Protein 1 (HP1) homolog Rhino, a marker of the dual-strand piRNA clusters, but are associated with HP1 itself, which provides piRNA-mediated transcriptional repression of the reporter genes. Unlike endogenous dual-strand piRNA clusters, the solo HeT-A promoter does not produce overlapping transcripts. In a telomeric context, however, bidirectional promoters of tandem HeT-A repeats provide read-through transcription of both genomic strands, followed by Rhi binding. These data indicate that Drosophila telomeres share properties of uni-strand and dual-strand piRNA clusters.
2016-12-07 | GSE78135 | GEO