Project description:Cells isolated from tibialis anterior muscle of mice model, and purified to Integrin7+; VCAM-1; Sca-1- and Lin- progenitors, and place onto culture dishes for 2-3 days before harvest for total RNA isolation
Project description:Transcriptional profiling of mouse myoblast cells comparing control vs. Mybbp1a knockdown. Stable clones of C2C12 cells harboring control or Mybbp1a-targeting shRNA were established and further pooled for analysis. Goal was to determine, based on the effects of Mybbp1a depletion on global gene expression, candidate downstream target genes of Mybbp1a, a putative transcriptional co-repressor.
Project description:In urodele amphibians, limb regeneration involves the dedifferentiation of muscle myotubes into single cells that may acquire pluripotent potential. We have employed small molecules (myoseverin and BIO) to attempt to reproduce this behavior in mammalian muscle culture. C2C12 myotubes derived from the C2C12 myoblast cell line were induced to undergo cellularization by myoseverin treatment, which destabilizes tubulin filaments. The GSK-3 inhibitor, BIO, was then used to induce dedifferentiation. Induce neuron formation; the cells were incubated with 250 nM reversine for 48 h, and neural induction media (DMEM/F12 supplemented with N2 (Invitrogen)) and 1.5 uM all-trans retinoic acid for 7 days. C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate (derived by 20 M myoseverin treatment for 48 h)
Project description:Transcriptional profiling of mouse myoblast cells comparing control vs. Mybbp1a knockdown. Stable clones of C2C12 cells harboring control or Mybbp1a-targeting shRNA were established and further pooled for analysis. Goal was to determine, based on the effects of Mybbp1a depletion on global gene expression, candidate downstream target genes of Mybbp1a, a putative transcriptional co-repressor. Two-condition experiment, control vs. Mybbp1a knockdown C2C12 cells (mixed stable clones). Biological replicates: 2.
Project description:The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. We used genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1 in C2C12 murine myoblast cells.
Project description:In urodele amphibians, limb regeneration involves the dedifferentiation of muscle myotubes into single cells that may acquire pluripotent potential. We have employed small molecules (myoseverin and BIO) to attempt to reproduce this behavior in mammalian muscle culture. C2C12 myotubes derived from the C2C12 myoblast cell line were induced to undergo cellularization by myoseverin treatment, which destabilizes tubulin filaments. The GSK-3 inhibitor, BIO, was then used to induce dedifferentiation. Induce neuron formation; the cells were incubated with 250 nM reversine for 48 h, and neural induction media (DMEM/F12 supplemented with N2 (Invitrogen)) and 1.5 uM all-trans retinoic acid for 7 days. C2C12 murine myoblast cell line and 48 h 10 uM BIO treated C2C12 cellulate (derived by 20 M myoseverin treatment for 48 h) C2C12 myoblasts were differentiated into myotubes with 2%horse serum in DMEM for 8 days (from 2-4 d, 10 uM AraC treatment was also used to kill any remaining myoblasts). Next, myotubes were cellularized by 20 uM myoseverin treatment for 48 h. 24 h after myoseverin treatment, myotubes were treated with 10 uM BIO for 2d.
Project description:The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. We used genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1 in C2C12 murine myoblast cells. C2C12 myoblast cells were seeded at a density of 2.5 x 10^5 cells / 10-cm plate one day prior to infection, and infected with a retrovirus containing a Flag-Msx1 construct for two consecutive days. The resulting cells were then crosslinked with formaldehyde, and DNA was enriched by chromatin immunoprecipitation (ChIP) with an anti-Flag antibody and analyzed by Solexa sequencing. Enriched regions were identified using a Poissonian background model, and were further compared to an additional background of sequences from a sample of Flag-immunoprecipitated DNA from C2C12 cells infected with an empty vector to determine enrichment. ChIP was performed using an antibody against the Flag epitope (Sigma M2, F3165).