By differing from the study of average cell profiles in a population, single-cell RNA sequencing has provided the opportunity to assess the transcriptomic composition of individual cells in a highly parallel manner. The single-cell RNA sequencing analysis of mononuclear cells from skeletal muscle, employing the Chromium Single Cell 3' solution from 10x Genomics' droplet-based technology, is detailed in this chapter. This protocol uncovers the identities of muscle resident cells, which provides a means for investigating the muscle stem cell niche in greater detail.

To support normal cellular functions, including the integrity of cellular membranes, metabolic processes, and the transmission of signals, appropriate lipid homeostasis is imperative. Skeletal muscle and adipose tissue play critical roles in the intricate process of lipid metabolism. During states of insufficient nutrition, adipose tissue, which stores triacylglycerides (TG), hydrolyzes these stores, releasing free fatty acids (FFAs). The skeletal muscle, requiring significant energy, utilizes lipids as oxidative substrates for energy production; however, excessive lipid metabolism can cause issues with muscle function. Lipid biogenesis and degradation cycles are dynamically influenced by physiological factors, and disrupted lipid metabolism is increasingly identified as a critical component of diseases including obesity and insulin resistance. Understanding the variety and changes in lipid composition is, thus, critical for adipose tissue and skeletal muscle. To explore diverse lipid classes in skeletal muscle and adipose tissue, we describe the method of multiple reaction monitoring profiling, utilizing lipid class and fatty acyl chain specific fragmentation. A detailed method for exploring acylcarnitine (AC), ceramide (Cer), cholesteryl ester (CE), diacylglyceride (DG), FFA, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and TG is presented. The characterization of lipid constituents in adipose and skeletal muscle tissues under diverse physiological circumstances may yield biomarkers and potential therapeutic avenues for addressing obesity-related illnesses.

Conserved across vertebrates, microRNAs (miRNAs) are small, non-coding RNA molecules, and they have critical roles in various biological processes. MicroRNAs (miRNAs) exert their influence on gene expression by both facilitating mRNA breakdown and hindering protein synthesis. The identification of muscle-specific microRNAs has given us a more comprehensive perspective of the molecular network involved in skeletal muscle function. We present a breakdown of methods frequently employed to analyze miRNA function in skeletal muscle.

One in 3,500 to 6,000 newborn boys develop Duchenne muscular dystrophy (DMD), a fatal condition linked to the X chromosome. A characteristic cause of the condition is an out-of-frame mutation specifically in the DMD gene's coding sequence. In exon skipping therapy, antisense oligonucleotides (ASOs), short, synthetic DNA-like molecules, are strategically used to excise problematic, mutated, or frame-shifting mRNA fragments, thus restoring the correct reading frame. The restored reading frame, in-frame, is guaranteed to produce a truncated, yet functional protein. The US Food and Drug Administration's recent approval of eteplirsen, golodirsen, and viltolarsen, ASOs, specifically phosphorodiamidate morpholino oligomers (PMOs), marks a milestone as the first ASO-based pharmaceuticals for Duchenne muscular dystrophy (DMD). Exon skipping, facilitated by ASOs, has been thoroughly examined in animal models. marker of protective immunity These models' DMD sequences are not identical to the human DMD sequence, which is problematic. Double mutant hDMD/Dmd-null mice, which solely incorporate the human DMD sequence and lack the mouse Dmd sequence entirely, are a viable solution to the presented issue. In this report, we detail intramuscular and intravenous administrations of an ASO targeting exon 51 skipping in hDMD/Dmd-null mice, alongside an in vivo assessment of its effectiveness.

AOs, or antisense oligonucleotides, have shown marked efficacy as a therapeutic intervention for genetic diseases, including Duchenne muscular dystrophy (DMD). By binding to a specific messenger RNA (mRNA), synthetic nucleic acids, AOs, can control the splicing of the RNA. In DMD, out-of-frame mutations are converted to in-frame transcripts via AO-mediated exon skipping. An exon skipping mechanism produces a protein that is both shortened and functional, akin to the milder form, Becker muscular dystrophy (BMD). Immunomagnetic beads Many prospective AO drugs, initially developed through laboratory research, are now being subjected to clinical trials due to a growing interest in this field. A critical aspect of proper efficacy assessment, prior to clinical trials, is the availability of an accurate and efficient in vitro method for testing AO drug candidates. In vitro AO drug screening procedures are significantly shaped by the type of cellular model utilized, and this model's choice demonstrably impacts the resulting data. Cell models previously utilized in screening for potential AO drug candidates, like primary muscle cell lines, demonstrate restricted proliferation and differentiation potential, and insufficient dystrophin production. Recently created immortalized DMD muscle cell lines successfully tackled this impediment, enabling accurate measurement of exon-skipping efficiency and the production of the dystrophin protein. The chapter explores a method used to measure the efficiency of skipping DMD exons 45-55, correlating this efficiency with dystrophin protein production in immortalized muscle cells derived from DMD patients. The potential for treating DMD gene patients, through exon skipping of exons 45-55, could reach approximately 47% of the affected population. Naturally occurring in-frame deletion mutations within exons 45 through 55 are associated with a milder, often asymptomatic, phenotype compared to shorter in-frame deletions in this segment of the gene. Subsequently, the skipping of exons 45 through 55 represents a hopeful therapeutic pathway, benefiting a wider array of Duchenne muscular dystrophy patients. This method affords an improved pre-trial examination of potential AO drugs targeting DMD, before their implementation in clinical studies.

Injury to skeletal muscle triggers the activation of satellite cells, which are adult stem cells responsible for muscle regeneration and growth. The process of clarifying the functional roles of intrinsic regulatory factors that control stem cell (SC) activity is partly hampered by the technological obstacles presented by in-vivo stem cell editing. Despite the substantial documentation of CRISPR/Cas9's genome-editing capabilities, its practical implementation within endogenous stem cells has seen limited exploration. Employing Cre-dependent Cas9 knock-in mice and AAV9-mediated sgRNA delivery, a recent study has produced a muscle-specific genome editing system for in vivo gene disruption in skeletal muscle cells. The above system allows for an illustration of efficient editing, achieved by following the step-by-step procedure shown here.

The CRISPR/Cas9 system possesses the capability to modify a target gene in all but a very few species, making it a powerful tool in genetic engineering. The creation of knockout or knock-in genes in laboratory animals now extends to species beyond the common mouse model. The Dystrophin gene's role in human Duchenne muscular dystrophy is apparent, but Dystrophin gene-mutated mice do not show the same extreme muscle degenerating characteristics as observed in humans. Differently, rats modified for a Dystrophin gene mutation using the CRISPR/Cas9 system demonstrate more pronounced phenotypic outcomes than mice. Rats with mutations in the dystrophin gene exhibit phenotypes that are more representative of the traits present in human DMD. Mice, when compared to rats, prove less effective models for studying human skeletal muscle diseases. learn more Employing the CRISPR/Cas9 system, we detail in this chapter a protocol for creating genetically modified rats through embryo microinjection.

MyoD, a transcription factor of the bHLH class and a key player in myogenic differentiation, demonstrates its potency by enabling fibroblasts to differentiate into muscle cells with its sustained presence. MyoD expression cycles in activated muscle stem cells throughout development (from developing to postnatal to adult) depending on conditions, such as their isolation in culture, their association with single muscle fibers, or their presence in muscle biopsies. A 3-hour oscillation period stands in stark contrast to the length of a typical cell cycle or circadian rhythm. Sustained MyoD expression, coupled with erratic MyoD oscillations, is a hallmark of stem cell myogenic differentiation. The oscillatory expression pattern of MyoD is dictated by the periodic expression of the bHLH transcription factor Hes1, which consistently represses MyoD's expression. Interference with the Hes1 oscillator's activity disrupts the sustained MyoD oscillations, causing a prolonged period of continuous MyoD expression. Maintaining activated muscle stem cells is crucial for muscle growth and repair, and this interference disrupts that process. Accordingly, the rhythmic variations in MyoD and Hes1 levels control the balance between the increase and transformation of muscle stem cells. Luciferase-based time-lapse imaging methodologies are presented for the monitoring of dynamic MyoD gene expression in myogenic cells.

The temporal regulation of physiology and behavior is orchestrated by the circadian clock. Diverse tissue growth, remodeling, and metabolic processes are heavily dependent on the cell-autonomous clock circuits specific to skeletal muscle. Recent breakthroughs unveil the inherent properties, intricate molecular controls, and physiological contributions of the molecular clock oscillators in both progenitor and mature myocytes of muscle tissue. A sensitive real-time monitoring approach, epitomized by a Period2 promoter-driven luciferase reporter knock-in mouse model, is critical for defining the muscle's intrinsic circadian clock, while different strategies have been applied to investigate clock functions in tissue explants or cell cultures.