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Transcriptome of mouse primary muscle cultures upon dystrophin knockdown by RNAi.

ABSTRACT: Dystrophin was knocked down in primary muscle cultures prepared from C57Bl/10 neonate mice using siRNA targeting dystrophin. Also in parallel, primary muscle cultures treated with siRNA targting luciferase were used as controls. The experiment was designed so that the same cell populations were used for both test and control conditions. This helped avoid the heterogeneity associated with the previous studies where test and control cell populations were non-identical. The experiment resulted in a clear transcriptome of dystrophin deficiency and demonstrated dystrophin as a major organizer of myogensis. Moreover, several interesting experimental targets were identified which can potentially open new lines of investigations.

ORGANISM(S): Mus musculus

PROVIDER: GSE20548 | GEO | 2010/12/21

SECONDARY ACCESSION(S): PRJNA125305

REPOSITORIES: GEO

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Transcriptome of mouse primary muscle cultures upon dystrophin knockdown by RNAi.

Project description:Dystrophin was knocked down in primary muscle cultures prepared from C57Bl/10 neonate mice using siRNA targeting dystrophin. Also in parallel, primary muscle cultures treated with siRNA targting luciferase were used as controls. The experiment was designed so that the same cell populations were used for both test and control conditions. This helped avoid the heterogeneity associated with the previous studies where test and control cell populations were non-identical. The experiment resulted in a clear transcriptome of dystrophin deficiency and demonstrated dystrophin as a major organizer of myogensis. Moreover, several interesting experimental targets were identified which can potentially open new lines of investigations. 18 primary muscle cell cultures were prepared on 4 occasions. 7 cultures were treated with siRNAs targeting dystrophin, 7 received siRNA targeting firefly GL2 luciferase (treatment controls) and 4 remained untreated (untreated controls). RNAs were extracted 48 hours after differentiation induction for expression profiling analysis.

2010-12-21 | E-GEOD-20548 | biostudies-arrayexpress

Histone deacetylase 4 represses dystrophin-glycoprotein (DGC) complex expression

Project description:Dysfunction of the dystrophin-glycoprotein complex (DGC) is a frequent cause of hereditary forms of muscular dystrophy. Although DGC function in maintaining skeletal muscle integrity has been well characterized, little is known about how the DGC complex is coordinately regulated at the transcriptional level. To test this hypothesis, we engineered HDAC4 stably overexpressing and control myotubes in an in vitro model of muscle differentiation. Here we present evidence that HDAC4, a neural activity-responsive histone deacetylase, is a critical transcriptional regulator of the DGC complex. We show that HDAC4 can repress multiple components of the DGC complex, including dystrophin and sarcoglycan family members in both cultured myotubes. To confirm this finding, the protein levels of core DGC complex members including dystrophin, sarcoglycan complex members, and additional dystrophin-associated proteins were evaluated in differentiated myotubes by western analysis. Keywords: genetic modification and cell type comparison of muscle cells

2008-03-25 | GSE10678 | GEO

Histone deacetylase 4 represses dystrophin-glycoprotein (DGC) complex expression

Project description:Dysfunction of the dystrophin-glycoprotein complex (DGC) is a frequent cause of hereditary forms of muscular dystrophy. Although DGC function in maintaining skeletal muscle integrity has been well characterized, little is known about how the DGC complex is coordinately regulated at the transcriptional level. To test this hypothesis, we engineered HDAC4 stably overexpressing and control myotubes in an in vitro model of muscle differentiation. Here we present evidence that HDAC4, a neural activity-responsive histone deacetylase, is a critical transcriptional regulator of the DGC complex. We show that HDAC4 can repress multiple components of the DGC complex, including dystrophin and sarcoglycan family members in both cultured myotubes. To confirm this finding, the protein levels of core DGC complex members including dystrophin, sarcoglycan complex members, and additional dystrophin-associated proteins were evaluated in differentiated myotubes by western analysis. C2C12 mouse myotubes were infected with either a HDAC4 expressing or control (Neo) retroviruses. After stable selection, myotubes were differentiated at 90% confluency in 2% horse serum (Hyclone) for 4 days. At this time point, RNA was extracted and the two types of cells compared in a microarray analysis.

2010-05-25 | E-GEOD-10678 | biostudies-arrayexpress

Transcription profiling of mouse dystrophin-deficient and dystrophin and utrophin double-deficient animalse crossed with mice with full-length hDMD genes

Project description:Crossing of hDMD mice that contain the full-length 2.3 Mb hDMD gene were crossed with dystrophin-deficient mdx mice and dystrophin and utrophin double-deficient mdx x utrn-/- mice resulted in a full rescue of the dystrophic features of these mice, as concluded from histological analysis. Analysis on Affymetrix gene chips demonstrated that also expression profiles of the dystrophic mice were normalized by crossing with transgenic hDMD mice. This confirms the full functionality of the hDMD transgene in mice. Experiment Overall Design: RNA from gastrocnemius muscle from individual mice was hybridized to Affymetrix U74Av2

2008-06-15 | E-GEOD-6790 | biostudies-arrayexpress

Transcription profiling of 16-week-old mdx mouse muscles to characterize transcriptional changes induced by dystrophin deficiency.

Project description:Despite over 3,000 articles published on dystrophin in the last 15 years, the reasons underlying the progression of the human disease, differential muscle involvement, and disparate phenotypes in different species are not understood. The present experiment employed a screen of 12,488 mRNAs in 16-wk-old mouse mdx muscle at a time when the skeletal muscle is avoiding severe dystrophic pathophysiology, despite the absence of a functional dystrophin protein. A number of transcripts whose levels differed between the mdx and human Duchenne muscular dystrophy were noted. A fourfold decrease in myostatin mRNA in the mdx muscle was noted. Differential upregulation of actin-related protein 2/3 (subunit 4), beta-thymosin, calponin, mast cell chymase, and guanidinoacetate methyltransferase mRNA in the more benign mdx was also observed. Transcripts for oxidative and glycolytic enzymes in mdx muscle were not downregulated. These discrepancies could provide candidates for salvage pathways that maintain skeletal muscle integrity in the absence of a functional dystrophin protein in mdx skeletal muscle.

2007-11-06 | E-GEOD-466 | biostudies-arrayexpress

Dystrophin Deficiency Derives Phenotype Switching in Vascular Smooth Muscle Cells

Project description:To investigate how dystrophin deficiency affects phenotype of vascular smooth muscle cells

2024-05-31 | GSE232219 | GEO

Dystrophin domains R4-15 and R20-23

Project description:Goal was to assess protein linkers between microtubules and dystrophin (specifically dystrophin regions R4-15 and R20-23). Two paired 10-plex TMT experiments were used to compare sixteen unique Dystrophin Glycoprotein Complex (or microtubule) enrichments (four genotypes, each with n=4) plus four pooled samples to allow comparison across the two individual 10-plex experiments for microtubules or DGC. A pair of 10-plex TMT(20 tags total) for the DGC enrichements, and a second pair of TMT (20 tags) for the MT enrichments. Samples are gastroc skeletal muscle from mouse.

2020-09-21 | PXD021088 | Pride

CRISPR/Cas9 editing of directly reprogrammed myogenic progenitors restores dystrophin expression in a dystrophic mouse model

Project description:Genetic mutations in dystrophin manifest in Duchenne muscular dystrophy (DMD), the most prevalent form of a genetically inherited muscle disease. Dystrophin is expressed in skeletal muscle stem cells and fibers, playing a critical role in maintaining skeletal muscle structure, regeneration and function. Here, we report on direct reprogramming of fibroblasts from the Dmdmdx mouse model into induced myogenic progenitor cells (Dmdmdx iMPCs) utilizing transient MyoD overexpression in concert with small molecule treatment. Dmdmdx iMPCs proliferate extensively in vitro and express canonical skeletal muscle stem and progenitor cell markers including Pax7, Sox8 and Myf5. Furthermore, Dmdmdx iMPCs readily give rise to highly contractile and multinucleated myofibers that express a suite of mature skeletal muscle genes however lack dystrophin expression. Utilizing an exon-skipping based approach with CRISPR/Cas9 we report on a genetic correction of the dystrophin mutation in Dmdmdx-iMPCs and subsequent restoration of dystrophin protein expression in vitro. Furthermore, engraftment of genetically-corrected Dmdmdx iMPCs into dystrophic limb muscles of Dmdmdx mice restored dystrophin expression in vivo. Collectively, our findings report on a novel in vitro stem cell-based model for DMD and further establish a new approach to treat this disease via a combination of direct cellular reprogramming, genome engineering and stem cell transplantation methods.

2021-12-15 | GSE164599 | GEO

Long-term maintenance of dystrophin expression and resistance to injury of skeletal muscle in gene-edited mice

Project description:Duchenne Muscular Dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome-editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, we subjected DEx44 DMD mice to systemic delivery of AAV9 expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. We showed that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection, satellite cells were unable to restore dystrophin expression due to restriction of gene editing components. Analysis of on-target and off-target gene editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.

2021-07-27 | GSE180771 | GEO

The DMD locus harbours multiple long non-coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms

Project description:The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (ncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of DMD symptomatic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD-specific lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by selective targeting and down-modulation of dystrophin promoter transcriptional activity.

2011-06-05 | GSE27068 | GEO

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