Myogenesis Methods and Protocols

Our understanding of the molecular and cellular mechanisms that control skeletal muscle development, regeneration, and adaptive responses to activity has increased dramatically in recent years, fostered by innovative techniques and approaches that are either specifically designed or adapted for research in skeletal muscle biology.  Myogenesis: Methods and Protocols presents detailed, step-by-step methods in the study of the molecular and cellular biology of skeletal muscle cells.  Protocols from different model systems including mammalian, avian, zebrafish, and invertebrate skeletal muscle are included in this volume.  Highlighted topics cover a wide range of interests and expertise including myogenic and stem cell isolation, investigation of models of exercise and disuse, viral vector delivery systems, calcium imaging, cell profiling, as well as protein-DNA and protein-protein interactions. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Isolation and Characterization of Human Fetal Myoblasts.- Skeletal Muscle Satellite Cells: Background and Methods for Isolation and Analysis in a Primary Culture System.- Isolation of Muscle Stem Cells by Fluorescence Activated Cell Sorting (FACS) Cytometry.- Mouse and Human Mesoangioblasts: Isolation and Characterization from Adult Skeletal Muscles.- Direct Electrical Stimulation of Myogenic Cultures for Analysis of Muscle Fiber Type Control.- Single Muscle-Fiber Isolation and Culture for Cellular, Molecular, Pharmacological, and Evolutionary Studies.- Somite Unit Chronometry to Analyze Teratogen Phase Specificity in the Paraxial Mesoderm.- Analysis of Skeletal Muscle Development in Drosophila.- Immunocytochemistry to Study Myogenesis in Zebrafish.- Immunofluorescent Localization of Proteins in C. elegans Muscle.- Resistance Loading and Signaling Assays for Oxidative Stress in Rodent Skeletal Muscle.- Analysis of Skeletal Muscle Hypertrophy in Models of Increased Loading.- Protein Overexpression in Skeletal Muscle Using Plasmid-Based Gene Transfer to Elucidate Mechanisms Controlling Fiber Size.- In Vivo Measurement of Muscle Protein Synthesis Rate Using the Flooding Dose Technique.- Recombinant Adeno-Associated Viral Vector Production and Purification.- Generation of Lentiviral Vectors for Use in Skeletal Muscle Research.- Generating Tamoxifen-Inducible Cre Alleles to Investigate Myogenesis in Mice.- Gene Profiling Studies in Skeletal Muscle by Quantitative Real-Time Polymerase Chain Reaction Assay.- Analysis of Lipid Profiles in Skeletal Muscles.- Proteomic Analysis of Dystrophic Muscle.- Detection of Calcium Release via Ryanodine Receptors.- Measurement of Calcium Release Due to Inositol Trisphosphate Receptors in Skeletal Muscle.- Detection of Calcium Sparks inIntact and Permeabilized Skeletal Muscle Fibers.- Analysis of Calcium Transients in Cardiac Myocytes and Assessment of the Sarcoplasmic Reticulum Ca²⁺-ATPase Contribution.- Analysis of Muscle Gene Transcription in Cultured Skeletal Muscle Cells.- Analysis of Fiber Type Differences in Reporter Gene Expression of b-Gal Transgenic Muscle.- Determination of Gene Promoter Activity in Skeletal Muscles In Vivo.- Determination of MiRNA Targets in Skeletal Muscle Cells.- shRNA-Mediated Gene Knockdown in Skeletal Muscle.- Detection of NF-kB Activity in Skeletal Muscle Cells by Electrophoretic Mobility Shift Analysis.- Isolation of Nuclei from Skeletal Muscle Satellite Cells and Myofibers for Use in Chromatin Immunoprecipitation Assays.- An Improved Restriction Enzyme Accessibility Assay (REAA) for Analyzing Changes in Chromatin Structure in Samples of Limited Cell Number.- ChIP Enriched in Silico Targets (ChEST), a ChIP on Chip Approach Applied to Analyzing Skeletal Muscle Genes.