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Bulk RNA-seq analysis of wildtype and MYF5/MYOD/MYF6 knockdown porcine embryo

ABSTRACT: Bulk RNA-seq analysis of wildtype and MYF5/MYOD/MYF6 knockdown porcine embryo

PROVIDER: PRJNA1216616 | ENA |

REPOSITORIES: ENA

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Bulk RNA-seq analysis of wildtype and MYF5/MYOD/MYF6 knockdown porcine embryo

Project description:The pig is an important animal model that is increasingly used for biomedical research. For example, pigs produced using somatic cell nuclear transfer (SCNT) technology are relevant for transplantation strategies either involving xenotransplantation or the development of human organs in pig for exotransplantation. Pigs, however, are less well characterized than other animal models such as rodents. In previous studies, we produced pigs that lacked skeletal muscle as this was the first step in humanizing this lineage, and we characterized these MYF5/MYOD/MYF6 knockdown embryos to E35. In the current studies, we have evaluated the development and myogenesis in mid-stage porcine embryos produced by SCNT (E41-E90). We comprehensively examined mid-stage MYF5/MYOD/MYF6 knockdown pigs produced by deleting MYF5/MYOD/MYF6 from porcine fibroblasts, cloning these fibroblasts, transferring the early embryos to surrogate gilts, and harvesting the resulting fetal pigs at defined gestational ages. Here, we observed that these genetic deletions resulted in a complete absence of skeletal muscle in these embryos and fetal pigs, an absence of ribs, vertebral abnormalities and an absence of a sternum. Importantly, we observed that these embryos failed to progress beyond E62 indicating embryonic lethality. In addition, we examined porcine myogenesis by evaluating wildtype comparators using gross anatomical, morphological, histological, molecular biological analyzes as well as magnetic resonance imaging of both WT and knockdown embryos and fetal pigs between the ages of E41-E90. We have determined that the knockdown phenotype in pigs is more severe than that observed in rodents as mice with this multiplexed deletion were lethal in the immediate postnatal period. These studies provide an important platform for engineering humanized muscle in gene edited pigs.

2025-01-28 | GSE288246 | GEO

MyoD, Myf5, myogenin, or hrGFP expressed in 2C5/7 myoD-/-;myf5-/- fibroblast cells

Project description:Gene expression changes induced by MyoD or Myf5 were examined in a double-knockout fibroblast cell line lacking endogenous functional myoD or myf5 genes. Use of this cell line precluded the possibility of auto- or cross-activation of endogenous myoD or myf5. Myogenin or hrGFP were expressed in parallel samples as controls. Following infection with retrovirus - expressing the relevant myogenic regulatory factor (MRF) from the viral LTR promoter and hrGFP through an IRES element in the same mRNA transcript - GFP+ cells were sorted by FACS and harvested for total RNA. Keywords: other

2005-09-07 | GSE3245 | GEO

Transcription profiling of mouse 2C5/7 myoD-/-;myf5-/- fibroblast cells expressing MyoD, Myf5, myogenin, or hrGFP

2008-06-12 | E-GEOD-3245 | biostudies-arrayexpress

Distinct activities of Myf5 and MyoD indicate sequential roles in skeletal muscle lineage specification and differentiation (ChIP-Seq)

Project description:In this work we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits PolII and robustly activates gene transcription. Chip-seq profiling of MyoD, Myf5, Histone H4 acetylation (H4Ac), and Pol II in MyoD-/-; Myf5-/- MEFs (M&M MEFs)

2016-03-01 | E-GEOD-75370 | biostudies-arrayexpress

Distinct activities of Myf5 and MyoD indicate sequential roles in skeletal muscle lineage specification and differentiation (RNA-Seq)

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Distinct activities of Myf5 and MyoD indicate sequential roles in skeletal muscle lineage specification and differentiation (RNA-Seq)

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Distinct activities of Myf5 and MyoD indicate sequential roles in skeletal muscle lineage specification and differentiation (ChIP-Seq)

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ChIP-seq of Active Histone Mark (H3K27ac) and Myogenic Regulatory Factors (MYF5 and MYOD) in Fusion Negative Rhabdomyosarcoma Cell Lines

Project description:We performed Chip-seq analysis of Myogenic Regulatory Transcription Factors (MYF5 and MYOD) in Fusion Negative Rhabdomyosarcoma cell lines. Endogenous MYF5/H3K27ac Chip-seq was performed in Rh18 cells and MYOD-H3K27ac Chip-seq was performed in RD cells, given that these cell lines express these proteins in a mutually-exclusive manner. Analysis revealed a common subset of enhancer and promoter regions bound by these transcription factors that are enriched for cell cycle regulation and embryonic muscle development pathways. Keywords: rhabdomyiosarcoma, Chip-seq, chromatin, Transcription factos, MYF5, MYOD

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Expression data from rhabdomyosarcoma cells expressing Myod and E-protein heterodimer and controls

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Transcription profiling of mouse embryoic fibroblasts expressing wild type MyoD or its non-acetylatable version MyoD RRR to investigate how MyoD acetylation may contribute to differential gene activation.

Project description:The transcription factor MyoD can coax na?e fibroblasts or otherwise committed cells to adopt the skeletal muscle phenotype by activating the muscle gene expression program. Activation of muscle gene expression occurs in quantal steps with not all the target genes of MyoD being activated at the same time. Some genes are induced in the initial phases, others at later stages despite the fact that MyoD is present throughout the differentiation process. MyoD is post-translationally modified by phosphorylation, ubiquitination, and acetylation. Here, we have employed a model system in which MyoD and its non-acetylatable version were inducibly expressed in mouse embryonic fibroblasts derived from mice to investigate how MyoD acetylation may contribute to differential gene activation. Experiment Overall Design: Mouse embryos fibroblasts (MEFs) obtained from MyoD-/-/Myf5-/- animals will be transduced with retroviruses expressing an estrogen receptor hormone binding domain fused to either mouse MyoD wild type (ER-MyoD wt) or MyoD bearing three point mutations at lysine residues 99, 102, and 104 that render it no longer acetylatable ( ER-MyoD RRR). A retrovirus without the ER-MyoD insert was also employed as negative control.</p> Aim 2. We will compare genome-wide expression profiling of MEFs transduced with either ER-MyoD wt or ER-MyoD RRR cultured in the presence of a medium that promotes skeletal muscle differentiation (DM) supplemented with beta-estradiol for 0, 6, 12, and 24 hours, respectively.

2007-11-15 | E-GEOD-6487 | biostudies-arrayexpress

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