myoblast transfection

BACKGROUND: Skeletal muscle is the largest tissue in the body, and it affects motion, metabolism and homeostasis. Skeletal muscle development comprises myoblast proliferation, fusion and differentiation to form myotubes, which subsequently form mature muscle fibres. This process is strictly regulated by a series of molecular networks. Increasing evidence has shown that noncoding RNAs, especially microRNAs (miRNAs), play vital roles in regulating skeletal muscle growth. Here, we showed that miR-668-3p is highly expressed in skeletal muscle...

A major challenge of engineering larger macroscale tissues in vitro is the limited diffusion of nutrients and oxygen to the interior. For skeletal muscle, this limitation results in millimeter scale outcomes to avoid necrosis. One method to address this constraint may be to vascularize in vitro -grown muscle tissue, to support nutrient (culture media) flow into the interior of the structure. In this exploratory study, we examine culture conditions that enable myogenic development and endothelial cell survival within tissue engineered 3D muscles...

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial regulatory roles in many biological processes, including the growth and development of skeletal muscle. miRNA-100-5p is often associated with tumor cell proliferation and migration. This study aimed to uncover the regulatory mechanism of miRNA-100-5p in myogenesis. In our study, we found that the miRNA-100-5p expression level was significantly higher in muscle tissue than in other tissues in pigs. Functionally, this study shows that miR-100-5p overexpression significantly promotes the proliferation and inhibits the differentiation of C2C12 myoblasts, whereas miR-100-5p inhibition results in the opposite effects...

Myogenesis, the progression of proliferating skeletal myoblasts to terminally differentiated myotubes, regulates thousands of target genes. Uninterrupted linear arrays of such genes are differentially associated with specific chromosomes, suggesting chromosome specific regulatory roles in myogenesis. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle, shares common features with normal muscle cells. We hypothesized that RMS and myogenic cells possess differences in chromosomal organization related to myogenic gene arrangement...

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p ) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo...

AIMS: Patients with ADP-ribose-acceptor hydrolase 3 ( ARH3 ) deficiency exhibit stress-induced childhood-onset neurodegeneration with ataxia and seizures (CONDSIAS). ARH3 degrades protein-linked poly(ADP- ribose) (PAR) synthesized by poly(ADP-ribose)polymerase (PARP)-1 during oxidative stress, leading to cleavage of the ADP-ribose linked to protein. ARH3 deficiency leads to excess accumulation of PAR, resulting in PAR-dependent cell death or parthanatos. Approximately one-third of patients with homozygous mutant ARH3 die from cardiac arrest, which has been described as neurogenic, suggesting that ARH3 may play an important role in maintaining myocardial function...

In recent years, an increasing number of studies have reported that long non-coding RNAs (lncRNAs) play essential regulatory roles in myogenic differentiation. In this study, a specific LncRNA XLOC_015548 (Lnc000280) was identified. However, little research has explored its mechanism of action by constructing XLOC_015548 gene editing cell models. In this study, relevant sequences were obtained according to the RNA-seq results. Subsequently, XLOC_015548 knockdown and over-expression lentiviral vectors were constructed, and the C2C12 myoblast cell line was transfected to prepare the XLOC_015548 gene-edited myoblast model...

Muscles that are injured or atrophied by aging undergo myogenic regeneration. Although myoblasts play a pivotal role in myogenic regeneration, their function is impaired with aging. MicroRNAs (miRNAs) are also involved in myogenic regeneration. MiRNA (miR)-1 and miR-133a are muscle-specific miRNAs that control the proliferation and differentiation of myoblasts. In this study, we determined whether miR-1 and miR-133a expression in myoblasts is altered with cellular senescence and involved in senescence-impaired myogenic differentiation...

At the neuromuscular junction (NMJ), DOK7 enhances the phosphorylation of muscle-specific kinase (MuSK) and induces clustering of acetylcholine receptors (AChRs). We identified a patient with congenital myasthenic syndrome (CMS) with two heteroallelic mutations in DOK7, c.653-1G>C in intron 5 and c.190G>A predicting p.G64R in the pleckstrin homology domain. iPS cells established from the patient (CMS-iPSCs) showed that c.653-1G>C caused in-frame skipping of exon 6 (120 bp) and frame-shifting activation of a cryptic splice site deleting seven nucleotides in exon 6...

TBX15 , which encodes a differentiation-related transcription factor, displays promoter-adjacent DNA hypermethylation in myoblasts and skeletal muscle (psoas) that is absent from non-expressing cells in other lineages. By whole-genome bisulfite sequencing (WGBS) and enzymatic methyl-seq (EM-seq), these hypermethylated regions were found to border both sides of a constitutively unmethylated promoter. To understand the functionality of this DNA hypermethylation, we cloned the differentially methylated sequences (DMRs) in CpG-free reporter vectors and tested them for promoter or enhancer activity upon transient transfection...

BACKGROUND: Monoclonal antibody (mAb) therapy is a promising antiviral intervention for Coronovirus disease (COVID-19) with a potential for both treatment and prophylaxis. However, a major barrier to implementing mAb therapies in clinical practice is the intricate nature of mAb preparation and delivery. Therefore, here, in a pre-clinical model, we explored the possibility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mAb delivery using a mAb-expressing encapsulated cell system...

C2C12 cells are widely used in the muscle field, as they differentiate easily into myotubes and show limited constraints to culture as compared to primary myoblasts. Both C2C12 and primary myoblasts are hard to transfect, which affects downstream experiments. More than 95% of the reports published since 2015 with C2C12 cells have used one gold standard transfectant (i.e., Lipofectamine®), although several studies have suggested less than 30% efficiency of this reagent. In parallel, the capacity of other commercial reagents to transfect muscle cells remains largely unknown...

Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein pulsed electric fields are used to transiently permeabilize the cell membrane, allowing enhanced transmembrane transport. By localizing the electric field over small portions of the cell membrane using nanostructured substrates, it is possible to increase considerably the gene electrotransfer efficiency while preserving cell viability. In this study, we expand the frontier of localized electroporation by designing an electrotransfer approach based on commercially available cell culture inserts with polyethylene-terephthalate (PET) porous substrate...

As an important downstream effector gene in the hippo signaling pathway, large tumor suppressor gene 2 (LATS2) is involved in cell proliferation and differentiation, organ size and tissue regeneration, and plays an important role in regulating the growth and development of animal muscles. The purpose of this study is to explore the temporal expression of bovine LATS2 gene, and determine the key transcription factors for regulating bovine LATS2 gene. The result showed that bovine LATS2 gene was highly expressed in liver and longissimus dorsi, and was up-regulated in infancy muscle...

The regulation of skeletal muscle growth and development in chicken is complex. MicroRNAs (miRNAs) have been found to play an important role in the process, and more research is needed to further understand the regulatory mechanism of miRNAs. In this study, leg muscles of Jinghai yellow chickens at 300 d with low body weight (slow-growing group) and high body weight (fast-growing group) were collected for miRNA sequencing (miRNA-seq) and Bioinformatics analysis revealed 12 differentially expressed miRNAs (DEMs) between the two groups...

1. Methyltransferase-like 21C (METTL21C) and insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) play important roles in the proliferation of chicken myoblasts. However, it remains unclear whether there is protein-protein interaction between METTL21C and IGF2BP1 to regulate proliferation of chicken myoblasts.2. In this study, the Igf2bp1 gene was amplified from cDNA of liver tissue of Lueyang black-bone chicken to construct the overexpression vector HA- Igf2bp1 . The HA- Igf2bp1 and Flag- Mettl21c vectors were individually transfected and co-transfected into HEK293T, respectively...

Estrogen is a female hormone that plays a role in various tissues, although the mechanism in skeletal muscle has not been fully clarified. We previously showed that systemic administration of estrogen for 10 weeks ameliorated decreased exercise endurance in ovariectomized mice. To assess whether a long-term and muscle-specific activation of estrogen signaling modulates muscle function, we constructed an expression plasmid for a constitutively active estrogen receptor α (caERα) under the control of muscle creatine kinase (Mck) gene promoter/enhancer...

Tetrandrine is well known to act as a calcium channel blocker. It is a potential candidate for a tumor chemotherapy drug without toxicity. Tetrandrine inhibits cancer cell proliferation and induces cell death through apoptosis and autophagy. As cancer patients usually experience complications with sarcopenia or muscle injury, we thus assessed the effects of tetrandrine on skeletal muscle cells. We report in this study that a low dose of tetrandrine (less than 5 μM) does not affect the proliferation of C2C12 myoblasts, but significantly inhibits myogenic differentiation...

Angiogenesis is the formation of new blood vessels from pre-existing vessels. Adequate oxygen transport and waste removal are necessary for tissue homeostasis. Restrictions in blood supply can lead to ischaemia which can contribute to disease pathology. Vascular endothelial growth factor (VEGF) is essential in angiogenesis and myogenesis, making it an ideal candidate for angiogenic and myogenic stimulation in muscle. We established C2C12 mouse myoblast cell lines which stably express elevated levels of (i) human VEGF-A and (ii) dual human FGF4-VEGF-A...

Stress urinary incontinence (SUI) and pelvic floor disorder (PFD) are common conditions with limited treatment options in women worldwide. Regenerative therapy to restore urethral striated and pelvic floor muscles represents a valuable therapeutic approach. We aim to determine the CRISPR interference-mediated gene silencing effect of the nonviral delivery of nuclease-deactivated dCas9 ribonucleoprotein (RNP) complex on muscle regeneration at the cellular and molecular level. We designed four myostatin ( MSTN )-targeting sgRNAs and transfected them into rat myoblast L6 cells together with the dCas9 protein...