Rather than investigating the average characteristics of the cells in a population, single-cell RNA sequencing has revolutionized the characterization of the transcriptomic profiles of individual cells in a highly parallel manner. Employing the Chromium Single Cell 3' solution from 10x Genomics, this chapter outlines the workflow for single-cell transcriptomic analysis of mononuclear cells isolated from skeletal muscle, using a droplet-based RNA-sequencing approach. This protocol enables the revelation of muscle-resident cell type identities, permitting a more in-depth analysis of the muscle stem cell niche.
Maintaining normal cellular functions, including membrane structural integrity, cell metabolism, and signal transduction, hinges upon the critical role of lipid homeostasis. Lipid metabolism significantly involves two key tissues: adipose tissue and skeletal muscle. Excessive lipids are stored in adipose tissue as triacylglycerides (TG), which are hydrolyzed to release free fatty acids (FFAs) during periods of insufficient nutrition. While lipids are crucial oxidative substrates for energy generation in the energy-demanding skeletal muscle, their excess can manifest as muscle dysfunction. Lipid cycles of biogenesis and degradation are subject to physiological control, while the malfunction of lipid metabolism is frequently linked to diseases like obesity and insulin resistance. Importantly, deciphering the range and shifts in lipid composition within adipose tissue and skeletal muscle is of significant importance. Multiple reaction monitoring profiling, leveraging lipid class and fatty acyl chain specific fragmentation, allows for an exploration of different lipid classes within the context of skeletal muscle and adipose tissue. We furnish a comprehensive approach for investigating acylcarnitine (AC), ceramide (Cer), cholesteryl ester (CE), diacylglyceride (DG), FFA, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), and TG through detailed analysis. Lipid composition analysis in adipose and skeletal muscle tissue across a range of physiological situations may establish reliable biomarkers and treatment targets for diseases related to obesity.
MicroRNAs (miRNAs), small, non-coding RNA molecules, demonstrate significant conservation in vertebrates, fundamentally impacting numerous biological processes. The fine-tuning of gene expression is accomplished by miRNAs through the dual mechanisms of mRNA decay acceleration and protein translation inhibition. By identifying muscle-specific microRNAs, our knowledge of the molecular network in skeletal muscle has been significantly enhanced. To understand miRNA function in skeletal muscle, we describe these frequently utilized procedures.
Newborn boys are impacted by Duchenne muscular dystrophy (DMD), a fatal X-linked condition, with an estimated frequency of 1 in 3,500 to 6,000 annually. The condition is generally caused by the presence of an out-of-frame mutation within the DNA sequence of the DMD gene. ASOs, short, synthetic DNA-like molecules, are a key component of exon skipping therapy, a novel approach that removes mutated or frame-shifting mRNA segments to restore the correct reading frame. The restored reading frame, in-frame, will generate a truncated, but still functional, protein. As the first ASO-based drugs for DMD, eteplirsen, golodirsen, and viltolarsen, all classified as phosphorodiamidate morpholino oligomers (PMOs), have been recently approved by the US Food and Drug Administration. Animal models have been employed for an extensive study of exon skipping, which is facilitated by ASOs. https://www.selleckchem.com/products/dup-697.html A noteworthy problem with these models is the variation observed between their DMD sequences and the human DMD sequence. Double mutant hDMD/Dmd-null mice, characterized by their exclusive human DMD sequence and absence of the mouse Dmd sequence, constitute a solution to this issue. Intramuscular and intravenous delivery methods of an ASO intended to skip exon 51 in hDMD/Dmd-null mice are detailed, coupled with an assessment of its functional efficacy observed directly within the living organism.
Genetic diseases like Duchenne muscular dystrophy (DMD) have shown promise for treatment using antisense oligonucleotides (AOs). AOs' capability as synthetic nucleic acids enables them to bind to and influence the splicing process of a targeted messenger RNA (mRNA). The mechanism of AO-mediated exon skipping alters out-of-frame mutations, typically observed in DMD, into in-frame transcripts. The exon skipping method causes the formation of a shortened, yet still functional protein, exhibiting similarities to the milder disease, Becker muscular dystrophy (BMD). Oral microbiome Potential AO medications, previously tested in laboratory settings, are experiencing a surge in interest, prompting their advancement to clinical trials. An accurate and efficient in vitro method for assessing AO drug candidates, preceding their introduction into clinical trials, is imperative for proper evaluation of efficacy. The initial step in in vitro AO drug screening is the selection of the cell model, a critical factor impacting the subsequent results of the analysis and the broader evaluation process. Cell models used in the past for evaluating potential AO drug candidates, exemplified by primary muscle cell lines, demonstrated restricted proliferative and differentiation capacity and insufficient dystrophin levels. 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. This chapter outlines a process to determine the efficiency of skipping DMD exons 45-55 and the resulting dystrophin protein production in immortalized muscle cells that originated from patients with DMD. The phenomenon of exon skipping in the DMD gene, affecting exons 45 through 55, is potentially applicable to 47 percent of patients with this condition. Furthermore, naturally occurring in-frame deletion mutations within exons 45-55 are linked to an asymptomatic or remarkably mild clinical presentation when contrasted with shorter in-frame deletions found within this genomic region. Subsequently, the skipping of exons 45 through 55 represents a hopeful therapeutic pathway, benefiting a wider array of Duchenne muscular dystrophy patients. The method described herein allows a more comprehensive examination of potential AO drugs for DMD, preceding their use in clinical trials.
The adult stem cells that contribute to the growth and regeneration of skeletal muscle are the satellite cells. The functional understanding of intrinsic regulatory factors controlling stem cell (SC) activity is hampered, in part, by the technical challenges of in-vivo stem cell editing. Extensive studies have confirmed the capabilities of CRISPR/Cas9 in genome editing, yet its use in endogenous stem cells has remained largely untested in practice. Through a recent investigation, a muscle-specific genome editing system was constructed by utilizing Cre-dependent Cas9 knock-in mice and AAV9-mediated sgRNA delivery to permit in vivo gene disruption within skeletal muscle cells. We'll detail the step-by-step process of efficient editing using the aforementioned system, 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. Laboratory animals, apart from mice, gain the ability to have knockout or knock-in genes created. Despite the involvement of the Dystrophin gene in human Duchenne muscular dystrophy, Dystrophin gene-mutated mice do not display the same degree of severe muscle degeneration as their human counterparts. Alternatively, Dystrophin gene mutant rats, generated via the CRISPR/Cas9 system, manifest more severe phenotypic presentations than mice. The phenotypes observed in dystrophin-deficient rats more closely reflect the characteristics of human DMD. The study of human skeletal muscle diseases finds a superior model in rats, as opposed to mice. Glaucoma medications The CRISPR/Cas9 system is utilized in a detailed protocol for generating gene-modified rats by microinjecting embryos, presented in this chapter.
MyoD's sustained presence as a bHLH transcription factor, a master regulator of myogenic differentiation, is all that is required to trigger the differentiation of fibroblasts into muscle cells. In developing, postnatal, and adult muscle, activated muscle stem cells exhibit oscillating MyoD expression levels, regardless of whether they are dissociated and cultured, bound to individual muscle fibers, or sampled from muscle biopsies. Oscillations typically last around 3 hours, a considerably briefer timeframe compared to the cell cycle or circadian rhythm. Sustained MyoD expression, coupled with erratic MyoD oscillations, is a hallmark of stem cell myogenic differentiation. MyoD's expression oscillates in accordance with the rhythmic expression of the bHLH transcription factor Hes1, which periodically hinders MyoD's activity. Removing the Hes1 oscillator's function negatively impacts the stable rhythm of MyoD oscillations, causing extended periods of continuous MyoD expression. This disruption impedes the maintenance of active muscle stem cells, leading to impaired muscle growth and repair. Hence, the oscillatory patterns of MyoD and Hes1 govern the equilibrium between the proliferation and differentiation 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. The growth, remodeling, and metabolic functions of various tissues are intricately linked to the cell-autonomous clock circuits present within the skeletal muscle. Recent research elucidates the intrinsic properties, molecular regulatory pathways, and physiological functions of the molecular clock oscillators within progenitor and mature myocytes, a crucial aspect of muscle biology. In the context of examining clock functions in tissue explants or cell culture systems, pinpointing the tissue-intrinsic circadian clock in muscle demands the sensitive, real-time monitoring capability of a Period2 promoter-driven luciferase reporter knock-in mouse model.