Myogenesis: Methods and Protocols (Methods in Molecular Biology, v798)

✍ Scribed by Joseph X. DiMario

• Publisher: Springer
• Year: 2011
• Tongue: English
• Leaves: 574
• Edition: 2012
• Category: Library

✦ Synopsis

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.   Comprehensive and authoritative, Myogenesis: Methods and Protocols serves as an invaluable, state-of-the-art resource for experienced and emerging scientists in basic research as well as clinical and regenerative medicine.

✦ Table of Contents

Myogenesis798_9781617793424_1617793426......Page 1
front-matter......Page 2
Myogenesis Methods and Protocols......Page 4
Preface......Page 6
Contents......Page 8
Contributors......Page 12
Part 1_Isolation and Analysis of Skeletal Muscle Precursor Cells......Page 18
1. Introduction......Page 19
2.1. Dissociation of Primary Human Fetal Skeletal Muscle Tissue (see Note 1)
......Page 21
2.2.2. Preparation of Sample for FACS......Page 22
2.3.2. Immunocyto- chemistry for In Vitro Fusion Assay......Page 23
3.1. Dissociation of Primary Human Fetal Skeletal Muscle Tissue......Page 24
3.2.1. Thawing of Cryopreserved Sample Prior to FACS......Page 25
3.2.2. Preparation of Sample for FACS......Page 26
3.2.3. Fluorescence-Activated Cell Sorting......Page 29
3.3.1. In Vitro Cell Culture......Page 30
3.3.2. Immunocyto- chemistry for In Vitro fusion Assay ( see Note 16)......Page 32
4. Notes......Page 33
References......Page 35
1. Introduction......Page 37
1.1. The Satellite Cell Is Defined by Its Niche......Page 38
1.2. Functional Satellite Cells Are Required Throughout Life......Page 39
1.3. Detection of Satellite Cell Progeny by Temporal Expression Patterns of Myogenic-Related Transcription Factors......Page 41
1.4. Classic and Contemporary Approaches for Satellite Cell Isolation......Page 42
2.1. Strain and Age of Animals......Page 44
2.4. Cell Yield, Choice of Culture Dish and Cell Seeding Density......Page 46
2.6. Plate Coating Matrices......Page 47
2.7. Fixation and Immunostaining......Page 48
3.2. General Equipment......Page 49
3.4. Cell Isolation and Culture Reagents......Page 50
4.1. Cell Isolation and Culture......Page 51
4.2. Cell Culture Fixation and Immunostaining......Page 54
5. Notes......Page 56
References......Page 64
1. Introduction......Page 69
2.1. Reagents......Page 70
3.1. Single Cell Suspension......Page 71
3.2. Cell Staining......Page 72
3.3. Performing FACS......Page 73
3.4. Downstream Applications......Page 76
4. Notes......Page 77
References......Page 80
1. Introduction......Page 81
2.1. Basic Materials......Page 82
2.3. Medium Recipes......Page 83
3. Methods......Page 84
3.1. FACS-Isolation of Adult Murine MABs......Page 85
3.4. Spontaneous Differentiation......Page 87
3.9. Long-Term Storage of Cell Batches......Page 89
4. Notes......Page 90
References......Page 91
1. Introduction......Page 93
2.1. Myoblast Isolation and Myotube Stimulation......Page 96
3.1. Myoblast Isolation......Page 97
3.3. Immunocyto­chemistry......Page 98
References......Page 99
1. Introduction......Page 101
2.1. Single-Fiber Isolation and Culture......Page 105
2.2. Electron Microscopy......Page 107
3.1. Mouse Muscle Dissection......Page 108
3.2. Muscle Cleaning......Page 109
3.5. Gravity Sedimentation......Page 110
3.6. Plating Fibers......Page 111
3.9. Isolating Fibers from Zebrafish......Page 112
4. Notes......Page 113
References......Page 117
1. Introduction......Page 119
2. Materials......Page 122
3.1. Focal Segmentation Defects Caused by Transient Exposure to BrdU at Low Concentration......Page 123
3.2. Inhibition of New Somite Formation Caused by Transient Exposure to BrdU at High Concentration......Page 127
3.3. General Conclusions and Discussion......Page 130
4. Notes......Page 132
References......Page 136
Part 2_Non-Mammalian Models of Myogenesis......Page 141
1. Introduction......Page 143
2. Materials......Page 147
3. Methods......Page 148
3.1.1. Collection and Fixation of Embryos......Page 149
3.1.2. Treatment of Embryos with Antibodies, and Detection......Page 150
3.2.1. Preparation and Fixation of Larval and Pupal Samples......Page 152
3.2.3. Mounting Larval/Pupal Samples for Photomicroscopy......Page 154
3.3.1. Dissection of Discs from the Larva......Page 155
3.3.2. RNA Probe Synthesis......Page 156
3.3.3. Imaginal Disc In Situ Hybridization......Page 157
3.4.1. Mounting and Sectioning Samples......Page 158
3.4.3. Alternate Protocol: Immunofluorescent Staining of Cryosections......Page 159
3.5.1. Fixation and Embedding of Samples in Paraffin......Page 160
3.5.3. Antibody Staining of Paraffin Sections......Page 161
4. Notes......Page 163
References......Page 167
1. Introduction......Page 169
2.2. Buffers and Reagents......Page 171
2.3. Antibodies......Page 172
3.2. Whole Mount Immuno­cytochemistry: Paraformaldehyde Fixed (Fig.  1)......Page 176
3.3. Whole Mount Immunocytochemistry: Carnoy’s Fixed......Page 178
3.4. Sectioned Immunocytochemistry (Fig.  2)......Page 179
4. Notes......Page 180
References......Page 183
1. Introduction......Page 187
2. Materials......Page 188
2.1. Solutions......Page 190
3.1. Collect Worms......Page 191
3.3. Constant Spring Fixation......Page 192
3.4. Brief Description of Immunostaining Using Either Nonet or Constant Spring Fixed Worms......Page 193
4. Notes......Page 194
References......Page 196
Part 3_Experimental Models and Analysis of Skeletal Muscle Exercise and Disuse......Page 199
1. Introduction......Page 201
2.2. Custom Rat Dynamometer......Page 204
2.3. Custom Dynamometer for the Mouse......Page 205
2.4. Electrical Stimulation......Page 206
2.7. 8-Hydroxy-2¢ - Deoxyguanosine (8-OHdG)
......Page 207
2.10. MnSOD and CuZnSOD......Page 208
3.2. Stimulation Parameters for Obtaining Maximal Contractions......Page 209
3.3. Preparing and Positioning the Mouse in the Dynamometer......Page 210
3.4. Repetitive Loading Parameters in Rat Muscles......Page 211
3.5. Loading Parameters in the Mouse......Page 212
3.6. Using Eccentric Contractions in Rat and Mouse Muscles......Page 213
3.8. Duty Cycle Parameters for SSC Loading......Page 214
3.9. Muscle Preparation for Measuring Oxidative Stress in Loaded Skeletal Muscle......Page 215
3.10. Determining the GSH/GSSG Ratio......Page 216
3.11. Hydrogen Peroxide (H 2 O 2) Levels......Page 217
3.12. 8-Hydroxy-2¢ - Deoxyguanosine (8-OHdG)
......Page 218
3.13. Lipid Peroxidation......Page 219
3.14. Catalase Activity......Page 220
3.15. MnSOD and CuZnSOD......Page 222
4. Notes......Page 223
References......Page 224
1. Introduction......Page 229
2.2. Fiber Cross-Sectional Area......Page 232
3.2. Fiber Cross-Sectional Area......Page 233
3.3. In Situ Contraction Testing......Page 236
3.4. Protein Concentration/Content......Page 238
4. Notes......Page 239
References......Page 243
1. Introduction......Page 247
2.3. Muscle Harvesting......Page 249
3.1. Plasmid DNA Preparation......Page 250
3.2. Surgery, Plasmid Injection, and Electroporation......Page 251
3.4. Immuno histochemistry......Page 252
4. Notes......Page 255
References......Page 258
1. Introduction......Page 261
2. Materials......Page 263
2.1. In Vivo Protein Synthesis Protocol......Page 264
2.2. Preparation of Tissue and Blood for Measurement of l -[4 3 H]-Phenylalanine Specific Radioactivity......Page 265
3.1. In Vivo Protein Synthesis Measurement......Page 266
3.2. Preparation of Tissue and Blood for Measurement of l -[4 3 H]-Phenylalanine Specific Radioactivity......Page 269
3.3. Calculations for Estimating Protein Synthesis Rates......Page 272
4. Notes......Page 273
References......Page 279
Part 4_Generation of Viral Vectors and Transgenic Mice......Page 281
1. Introduction......Page 283
2.1. Recombinant AAV Vector Production......Page 285
2.3. Titer Determination and Quality Control......Page 287
3.1. Recombinant AAV Vector Production......Page 288
3.2. Recombinant AAV Purification......Page 289
3.3. Titer Determination and Quality Control......Page 290
4. Notes......Page 293
References......Page 298
1. Introduction......Page 301
2.2. Lentivirus Production......Page 304
3.1. Production of Lentivirus by Calcium Chloride Transfection ( see Note 2)......Page 305
3.2. Myoblast Transduction......Page 306
4. Notes......Page 307
References......Page 310
1. Introduction......Page 313
2. Materials......Page 314
3.1. Confirm the Ordered BAC......Page 315
3.3. Prepare Oligos and pL253 Vector to Capture Desired Genomic Region from the BAC......Page 316
3.5. Screen for Recombinants......Page 318
3.7. Screen for Recombinants......Page 319
3.9. Additional Steps......Page 320
4. Notes......Page 321
References......Page 323
Part 5_Muscle Profiling......Page 325
1. Introduction......Page 327
2.1. Cell Culture......Page 328
2.2.5. Primers......Page 329
3.2. Extraction of Total RNA from Cultured Myoblasts/Myotubes......Page 330
3.4. Evaluating RNA Quality......Page 331
3.6.1. Designing Primers......Page 333
3.6.2. Setting Up the Reaction......Page 336
4. Notes......Page 337
References......Page 339
1. Introduction......Page 341
2. Materials......Page 344
2.2. Analysis of Triacylglycerols and Total Phospholipids......Page 345
2.3. Analysis of Triacylglycerols and Individual Phospholipids......Page 348
3.1. Gas Chromatography......Page 349
3.2.1. Lipid Extraction and Separation......Page 350
3.2.2. Preparation and Analysis of Methyl Esters and Dimethyl Acetals......Page 354
3.2.3. Calculations......Page 355
3.3.1. Establishing the Separation Pattern of Phospholipids in 2D TLC......Page 357
3.3.2. Lipid Extraction and Separation......Page 360
3.3.3. Preparation and Analysis of Methyl Esters and Dimethyl Acetals......Page 363
3.3.4. Calculations......Page 364
4. Notes......Page 366
References......Page 370
1. Introduction......Page 373
2.1. Equipment......Page 374
2.2.1. Preparation of Crude Skeletal Muscle Extracts......Page 375
2.2.5. Reduction and Alkylation of Silver-Stained Proteins......Page 376
3.1. Preparation of Crude Muscle Extracts......Page 377
3.4. Isoelectric Focusing......Page 378
3.6. Protein Visualization Using Silver Staining......Page 379
3.7. Image Analysis of Protein Spot Patterns......Page 380
3.10. Reduce and Alkylate Preparative Silver-Stained Spots......Page 381
3.12. Electrospray Ionization-Mass Spectrometric Analysis......Page 382
4. Notes......Page 383
References......Page 384
Part 6_Experimental Approaches in Calcium Imaging of Skeletal Muscle......Page 387
1. Introduction......Page 389
2.1. Immuno fluorescent /Immuno histochemical Localization of RyR Isoforms......Page 390
2.2. Determination of RyR Protein Levels Using ( 3 H)Ryanodine-Binding Assay......Page 391
2.3. Detection of Ca 2+ Transients Mediated by RyRs ( see Note 2)......Page 392
3.2. Immuno­fluorescent/Immuno histochemical Detection of RyRs in Tissues ( see Note 7)......Page 393
3.5. Detection of Ca 2+ Transients Mediated by RyRs ( see Notes 2 and 13)......Page 394
4. Notes......Page 395
References......Page 397
1. Introduction......Page 399
2.1. Cultures of Myoblasts from Neonatal Skeletal Muscle......Page 400
3.1. Cultures of Myoblasts from Neonatal Skeletal Muscle......Page 401
3.3. Isolation of Adult Muscle Fibers from Mice......Page 402
3.4. Calcium Signal Measurement......Page 403
4. Notes......Page 406
References......Page 408
1. Introduction......Page 411
2.1. FDB Muscle Fiber Isolation......Page 413
2.4. Ca 2+ Spark Imaging of Permeabilized EDL Fibers......Page 414
3.1. FDB Muscle Fiber Isolation......Page 415
3.3. Ca 2+ Spark Imaging of Intact FDB Fibers......Page 417
3.4. Ca 2+ Spark Imaging of Permeabilized EDL Fibers......Page 418
3.5. Analysis of Ca 2+ Sparks Imaging from Isolated Muscle Fibers......Page 419
4. Notes......Page 420
References......Page 424
1. Introduction......Page 427
2.1. Primary Cell Cultures (Neonatal Rat Cardiac Myocytes)......Page 429
2.2. Calcium Transients......Page 431
3.1. Isolation of Neonatal Rat Cardiac Myocytes (Primary Cell Cultures ( 8))......Page 432
3.2. Calcium Transients Measurements......Page 433
3.3. Contribution of SERCA2 to Ca 2+ Transients in Cardiac Myocytes......Page 434
4. Notes......Page 435
References......Page 436
Part 7_Analysis of Gene Promoter Transcriptional Activity......Page 439
1. Introduction......Page 441
2.1. Identifying Regulatory Regions and Control Elements in Muscle Genes......Page 444
3.1. Identifying Regulatory Regions and Control Elements in Muscle Genes......Page 445
3.3. Identifying Transcription Factor Interactions......Page 446
3.4. Focus on Chromatin Immunoprecipitation......Page 447
3.5. Focus on Selective Enrichment of Transcription Factors for Identification by Quantitative Proteomics......Page 450
4. Notes......Page 453
References......Page 455
1. Introduction......Page 461
2.1.2. Cryofreezing with Fixation and Cryopreservation (Pretreatment Step)......Page 465
2.4. b -gal Staining......Page 466
3.1.1. Cryofreezing (Without Fixation)......Page 467
3.2. Preparation of Monoclonal Antibodies......Page 469
3.3. Fiber-Type Staining......Page 470
3.4. b -gal Staining......Page 471
4. Notes......Page 472
References......Page 474
1. Introduction......Page 477
2.1. Gene Promoter Reporter Plasmid Preparation......Page 479
3.1. Gene Promoter Reporter Plasmid Preparation......Page 480
3.1.2. Determining Plasmid Concentrations for Injections......Page 481
3.3. Luciferase Activity......Page 482
4. Notes......Page 484
References......Page 488
Part 8_RNA-Mediated Gene Regulation......Page 489
1. Introduction......Page 491
2.1.2. Quantitative RT-PCR......Page 495
2.3. Overexpression and Knockdown
of Muscle miRNAs in Cell Lines......Page 496
3.1. Detecting the Expression of Muscle miRNAs by Northern Blot and Quantitative RT-PCR Analyses......Page 497
3.2. Studying the Regulation of Muscle miRNAs on Their Targets by Luciferase Reporter Assays......Page 499
3.3. Overexpression and Knockdown of Muscle miRNAs in Cell Lines......Page 501
4. Notes......Page 503
References......Page 505
1. Introduction......Page 507
2.2. In Vivo Experiments......Page 509
3.1. In Vivo Electrotransfer......Page 510
3.2. Fluorescence Data Acquisition and Analysis......Page 511
4. Conclusion......Page 512
5. Notes......Page 515
References......Page 516
Part 9_Analysis of Protein-DNA Interactions......Page 519
1. Introduction......Page 521
2.3. Electrophoretic Mobility Shift Assay......Page 522
3.1. Preparation of Nuclear Extracts from Cell Pellets......Page 523
3.2. Preparation of Nuclear Extracts from Skeletal Muscle Tissue......Page 525
3.3. Generating a Radiolabeled Probe for Detecting NF- k B Binding by EMSA......Page 526
3.4.2. Preparation of Protein: DNA-Binding Reactions for Nuclear Extracts Isolated from Cellular Pellets......Page 527
3.4.5. Loading and Running the Samples......Page 528
3.5.1. Generate “Cold” Nonradiolabeled NF- k B-Specific Probe......Page 529
3.5.3. Supershift Analysis......Page 530
4. Notes......Page 531
References......Page 532
1. Introduction......Page 533
2.2. Separation of Satellite Cells and Myofibers......Page 535
3.1. Isolation and Digestion of Tissue......Page 537
3.2. Separation of Satellite Cells and Myofibers ( see Note 3)......Page 539
3.4. Examples of Results Achieved with Isolated Satellite Cell, Myofiber, and Liver (Control) Nuclei......Page 541
4. Notes......Page 543
References......Page 545
1. Introduction......Page 547
2.1. Isolation of Nuclei......Page 549
3.1. Isolation of Nuclei......Page 550
3.2. Restriction Enzyme Digestion......Page 551
3.3. Preparation of Linker and Ligation of Linker to Cleaved DNA......Page 552
3.4. PCR Detection of Cleaved DNA......Page 553
3.5. Detection of Changes in Restriction Enzyme Accessibility in as Few as 1,000 Cells......Page 554
4. Notes......Page 556
References......Page 557
1. Introduction......Page 559
2.1. Cross-linking and ChIP Components......Page 560
2.2. Purification and Amplification of Immuno­precipitated DNA......Page 561
3.1. In Silico Prediction of CRMs and Generation of Computed CRM Array......Page 562
3.2. ChIP from Drosophila Embryos......Page 564
3.3. DNA Amplification, Probe Synthesis, and Hybridization......Page 565
4. Notes......Page 566
References......Page 569
Index......Page 571