دانلود کتاب the MYC GENE : methods and protocols.

فهرست مطالب :

Preface
Acknowledgments
Contents
Contributors
Chapter 1: An ``-omycs´´ Toolbox to Work with MYC
References
Chapter 2: Methods of Expression, Purification, and Preparation of the c-Myc b-HLH-LZ for Its Biophysical Characterization
1 Introduction
2 Materials
2.1 Expression of c-Myc b-HLH-LZ Domain
2.2 Purification of the c-Myc b-HLH-LZ
3 Methods
3.1 Expression of the c-Myc b-HLH-LZ Domain
3.2 Purification of the c-Myc b-HLH-LZ
4 Notes
References
Chapter 3: Biophysical and Structural Methods to Study the bHLHZip Region of Human c-MYC
1 Introduction
2 Materials
2.1 Expression of the c-MYC bHLHZip and c-MYC:MAX bHLHZip Complex Constructs
2.2 Purification of c-MYC bHLHZip Protein and c-MYC:MAX bHLHZip Complexes
2.3 NMR Studies
2.4 Crystallization Studies
3 Methods
3.1 Expression of c-MYC bHLHZip and c-MYC:MAX bHLHZip Constructs in Rich or in Minimal Media
3.2 Purification of Cleavable His-Tag c-MYC bHLHZip Protein
3.3 Purification of c-MYC:MAX bHLHZip Complex Constructs
3.4 Production of Selectively Labeled c-MYC:MAX bHLHZip Complexes for NMR Studies
3.5 NMR Protocols to Study c-MYC bHLHZip by Itself and c-MYC:MAX bHLHZip Complex
3.6 Crystallization Protocol for c-MYC:MAX bHLHZip Complex
4 Notes
References
Chapter 4: Identifying and Validating MYC:Protein Interactors in Pursuit of Novel Anti-MYC Therapies
1 Introduction
1.1 Identifying and Validating MYC Interactors
1.2 BioID
1.3 Validation of BioID Results
2 Materials
2.1 Components of BioID
2.2 Components of In Vitro GST-Pull-Down Assay
2.3 Components of Proximity Ligation Assay
2.4 Components of Biolayer Interferometry
3 Methods
3.1 MYC-BioID (See Notes 1-3)
3.1.1 Preparation of Cells for BioID
3.1.2 Purification of Biotinylated Proteins
3.1.3 Mass Spectrometry Analysis
3.1.4 High-Confidence Interactome Network Analysis
3.2 In Vitro GST-Pull-Down Assay
3.2.1 Recombinant GST-MYC Purification
3.2.2 GST-Pull-Down
3.3 Proximity Ligation Assay
3.3.1 Immunofluorescence
3.3.2 Proximity Ligation Assay (Duolink In Situ Red Starter Kit Mouse/Rabbit)
3.4 BLI Performed on Octet System
3.4.1 Biotinylated MYC Sample Preparation
3.4.2 Running a Kinetic Experiment
4 Notes
References
Chapter 5: Detection of Post-translational Modifications on MYC
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 Antibody-Bead Conjugation
2.3 Immunoprecipitation
2.4 Western-Blot Analysis
2.5 Primary and Secondary Antibodies
2.6 Cell Transfection
2.7 Cell Ubiquitination and SUMOylation Assays
2.8 Formalin-Fixed Paraffin-Embedded Tissue Staining
3 Methods
3.1 Antibody to Bead Conjugation
3.2 Immunoprecipitation for Detection of c-Myc Serine 62 Phosphorylation
3.3 Detection of c-Myc Phosphorylation by Commercially Available Phospho-Specific Antibodies
3.4 Detection of c-Myc Ubiquitination in Cells
3.5 Detection of c-Myc SUMOylation in Cells
3.6 Immunofluorescence Staining of Formalin-Fixed Paraffin-Embedded Tissue
3.7 Detection of c-Myc in Formalin-Fixed Paraffin-Embedded Tissue
4 Notes
References
Chapter 6: MYC Analysis in Cancer and Evolution
1 Introduction
2 Materials
3 Methods
3.1 Preparation and Cultivation of Avian Embryo Fibroblasts
3.2 DNA Transfection of MYC-Carrying Retroviral Vector Plasmids
3.3 Focus Assay of MYC-Transformed Cells
3.4 Testing MYC Inhibitors by Agar Colony Assay and Cell Proliferation Measurement
3.5 MYC-Protein Interactions Monitored by Protein Pull-Down Analysis
3.5.1 Detection by Radioactive Protein Labeling
3.5.2 Detection by Immunoblotting Using ECL
3.6 MYC Protein Interactions Monitored by Co-immunoprecipitation (CoIP) Analysis
3.6.1 Detection by Radioactive Protein Labeling and Fluorography
3.6.2 Detection by Immunoblotting Using ECL
3.7 Analysis of MYC Expression by In Situ Hybridization in Hydra
3.8 Analysis of MYC Expression by Northern Analysis
3.9 Protein-DNA Interaction Analysis in Hydra by Chromatin Immunoprecipitation (ChIP)
3.10 MYC-DNA Interaction Analysis by Electrophoretic Mobility Shift Assay (EMSA)
4 Notes
References
Chapter 7: Genome-Wide Analysis of c-MYC-Regulated mRNAs and miRNAs and c-MYC DNA-Binding by Next-Generation Sequencing
1 Introduction
2 Materials
2.1 RNA-Seq
2.2 miRNA-Seq
2.3 ChIP-Seq
2.3.1 ChIP
2.3.2 ChIP-Seq Library Generation
3 Methods
3.1 RNA-Seq
3.1.1 mRNA Library Preparation
Quality and Quantity Control of Total RNA Input
mRNA Purification and Fragmentation
RNA Bead Plate Preparation, Washing, and Elution
Generation/Incubation of RNA Fragmentation Plate (RFP)
First Strand cDNA Synthesis
Second Strand Synthesis
cDNA Cleanup
End Repair
3′-end Adenylation
Adapter Ligation
DNA Fragment Enrichment
3.1.2 Library Quality Control
3.1.3 mRNA Library Sequencing
3.2 miRNA-Seq
3.2.1 miRNA Library Preparation
3′-Adapter Ligation
5′-Adapter Ligation
Sample Electrophoresis and RNA Gel Extraction
Reverse Transcription and Amplification
Template Preparation
PCR
miRNA Library Purification
Recovery of miRNA Library from Gel
3.2.2 miRNA Library Validation
3.2.3 miRNA Library Sequencing
3.3 ChIP-Seq
3.3.1 ChIP
Experimental Design of ChIP for Subsequent NGS Analysis
Pre-blocking of Sepharose Beads
Cross-Linking and Sonication
Immunoprecipitation
Cross-Link Removal and Purification
Quality and Quantity Control of Immunoprecipitated DNA
3.3.2 ChIP-Seq Library
ChIP-Seq Library Generation
End Repair
Addition of ``A´´ Bases to the 3′ End of the DNA Fragments
Adapter to DNA Fragment Ligation
Size Selection of the Library
Enrichment of Adapter-Modified DNA Fragments by PCR
ChIP-Seq Library Validation
3.3.3 Library Sequencing
3.4 Bioinformatics Analyses of NGS Results
3.4.1 ChIP-Seq Bioinformatics
3.4.2 mRNA-Seq Bioinformatics
3.4.3 miR-Seq Bioinformatics
3.5 Validation of NGS Results
3.5.1 qPCR Validation of c-MYC-Regulated Transcripts
3.5.2 qPCR Validation of c-MYC-Regulated miRNAs
3.5.3 qChIP Validation of Occupancy by c-MYC
4 Notes
References
Chapter 8: Analysis of Myc Chromatin Binding by Calibrated ChIP-Seq Approach
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers
2.3 Equipment
2.4 Software
3 Methods
3.1 Cell Harvest
3.2 Chromatin Immunoprecipitation
3.3 DNA Purification
3.4 Library Preparation and Sequencing
3.5 Data Analysis
3.6 Peak Calling
3.7 GSEA for Myc-Binding Sites
3.8 Metagenome Analysis
3.9 Intersecting RNA-Seq and ChIP-Seq Data
4 Notes
References
Chapter 9: A High-Throughput Chromatin Immunoprecipitation Sequencing Approach to Study the Role of MYC on the Epigenetic Land...
1 Introduction
2 Materials
2.1 Reagents
2.2 Buffers
2.3 Equipment
2.4 Software and Bioinformatic Tools
3 Methods
3.1 Cell Harvest and Fixation
3.2 Cell Lysis and Chromatin Sonication
3.3 Chromatin Immunoprecipitation and De-crosslinking
3.4 DNA Purification with SPRI Beads and HT-ChIP Validation by qPCR
3.5 Library Preparation
3.6 Reads Quality Control, Mapping, and Visualization
3.7 Peak Calling
4 Notes
References
Chapter 10: Methods for Determining Myc-Induced Apoptosis
1 Introduction
2 Materials
2.1 In Vitro Assays
2.1.1 Cell Culture and Microscopy
2.1.2 Flow Cytometry for Annexin V
2.1.3 Western Blotting for Cleaved Caspase 3
2.2 In Vivo Assays
2.2.1 Histology
2.2.2 Haematoxylin and Eosin (H&E) Staining
2.2.3 TUNEL Staining
2.2.4 Immunohistochemistry for Cleaved Caspase 3
2.2.5 Western Blotting for Cleaved Caspase 3
2.2.6 RNA Analysis
3 Methods
3.1 Phase Contrast Microscopy
3.2 Detection of Exposed Annexin V by Flow Cytometry
3.3 Western Blotting for Cleaved Caspase 3
3.4 Haematoxylin and Eosin (H&E) Stain
3.5 Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL)
3.6 Immunohistochemistry for Activated Caspase 3
3.7 Western Blotting of Tissue Samples for Cleaved Caspase 3
3.8 Isolation of RNA and Determination of RNA Expression of Selected Proapoptotic Proteins
3.9 Conclusions
4 Notes
References
Chapter 11: Measuring MYC-Mediated Metabolism in Tumorigenesis
1 Introduction
2 Materials
2.1 Polar Metabolite Extraction
2.2 Lipid Extraction
3 Methods
3.1 Extraction of Polar Metabolites for LC-MS/MS Analysis
3.2 Extraction of Nonpolar Lipids for LC-MS/MS Analysis
4 Notes
References
Chapter 12: Methods to Study Myc-Regulated Cellular Senescence: An Update
1 Introduction
2 Materials
2.1 Reagents and Solutions for SA-β-gal Detection In Situ
2.2 Reagents and Solutions for EdU Labeling
2.3 Reagents and Solutions for Phalloidin and DAPI Staining
2.4 Reagents and Solutions for SA-β-gal Detection in Cell Lysates
2.5 Reagents and Solutions for Sudan Black B (SBB) Analogue GL13 Detection in Tissue Sections
3 Methods
3.1 Simultaneous Detection of Multiple Senescence Markers In Situ
3.2 Fluorogenic SA-β-gal Assay (MUG Assay)
3.3 Detection of Senescent Cells in Formalin-Fixed Paraffin-Embedded Tissues with Sudan Black B (SBB) Analogue GL13
4 Notes
References
Chapter 13: Examining Myc-Dependent Translation Changes in Cellular Homeostasis and Cancer
1 Introduction
2 Materials
2.1 35S Metabolic Labeling in Cultured Cells
2.2 O-Propargyl-Puromycin (OP-Puro) Incorporation In Vivo
2.3 Investigating Specific Alterations in mRNA Translation by Fractionating Polysome-Associated mRNAs
2.4 Toeprint Assay to Visualize the Position of Translating Ribosomes
2.5 Measuring Translation Initiation and Start Codon Usage Using a Reporter Assay
3 Methods
3.1 35S Metabolic Labeling in Cultured Cells
3.2 O-Propargyl-Puromycin (OP-Puro) Incorporation In Vivo
3.3 Investigating Specific Alterations in mRNA Translation by Fractionating Polysome-Associated mRNAs
3.4 Toeprint Assay to Visualize the Position of Translating Ribosomes
3.5 Measuring Translation Initiation and Start Codon Usage Using a Reporter Assay
4 Notes
References
Chapter 14: A Versatile In Vivo System to Study Myc in Cell Reprogramming
1 Introduction
2 Materials
2.1 AAV Vectors (see Note 2)
2.2 Mice and Injection Material
2.3 Immunochemistry Material
2.4 iPS Cells Isolation
3 Methods
3.1 AAV Vector Injection
3.2 Mouse Monitoring
3.3 Histopathological Analysis of Teratomas
3.3.1 Hematoxylin and Eosin Staining
3.3.2 Immunohistochemistry
3.4 Histopathological Evaluation of Organs
3.5 In Vivo Induced Pluripotent Stem Cells Isolation
3.5.1 iPS Cells Isolation from Teratomas
3.5.2 iPS Cells Isolation from Peripheral Blood
3.5.3 iPS Cells Isolation from Bone Marrow
4 Notes
References
Chapter 15: Using Flow Cytometry to Study Myc´s Role in Shaping the Tumor Immune Microenvironment
1 Introduction
2 Materials
2.1 Mouse Necropsy and Sample Collection
2.2 Reagents for Tumor Single-Cell Preparation
2.3 Reagents for T Cell Activation
2.4 Reagents and Antibodies for Cell Surface and Intracellular Antigen Staining
3 Methods
3.1 Mouse Necropsy and Sample Collection
3.2 Dissociation of Mouse Tumors and Single-Cell Suspension Preparation
3.3 T Cell Activation
3.4 Surface and Intracellular Flow Cytometry Staining
3.4.1 Dead Cell Labeling
3.4.2 Surface Antigen Staining
3.4.3 Intracellular Antigen Staining
Transcription Factor Staining Protocol
Intracellular Cytokines Staining Protocol
4 Notes
References
Chapter 16: Generation of a Tetracycline Regulated Mouse Model of MYC-Induced T-Cell Acute Lymphoblastic Leukemia
1 Introduction
2 Materials
2.1 Generation of the Tet-O-MYC and EμSRα-tTA Constructs
2.2 In Vitro Validation of Conditional Expression of the Tet-O-MYC Construct
2.3 Genotypic Analysis of Founders and Progeny
2.4 Identification of Founders with Conditional Gene Expression
2.5 Mouse Necroscopy and Sample Collection
2.6 FACS Analysis of Primary Tumor to Identify the Phenotype of Cells Comprising the Tumor
2.7 Generating Cell Lines from Primary Tumor Tissue
2.8 Evaluating Conditional Expression of Tet System In Vivo
2.9 In Vivo Tumor Transplantation Studies
3 Methods
3.1 Generation of the Tet-O-MYC and EμSRα-tTA Constructs
3.1.1 Preparation of the Tet-O-MYC Transgenic Construct
3.1.2 Preparation of the EμSRα-tTA Transgenic Construct
3.1.3 To Prepare the Transgenic Constructs for Microinjection
3.2 In Vitro Validation of Conditional Expression of the Tet-O-MYC Construct
3.3 Genotypic Analysis of Founders and Progeny
3.4 Identification of Founders with Conditional Gene Expression
3.5 Mouse Necropsy and Sample Collection
3.6 FACS Analysis of Primary Tumor to Identify the Phenotype of Cells Comprising the Tumor
3.7 Generating Cell Lines from Primary Tumor Tissue
3.8 Evaluating Conditional Expression of Tet System In Vivo
3.9 In Vivo Tumor Transplantation Studies
4 Notes
References
Chapter 17: Chromogenic In Situ Hybridization (CISH) as a Method for Detection of C-Myc Amplification in Formalin-Fixed Paraff...
1 Introduction
2 Materials
3 Methods
3.1 CISH Procedure (See Also Note 1)
3.2 Evaluation of Results
4 Notes
References
Chapter 18: MYC Copy Number Detection in Clinical Samples Using a Digital DNA-Hybridization and Detection Method
1 Introduction
1.1 Hybridization
1.2 Purification
1.3 Data Collection
1.4 Data Analysis
2 Materials
2.1 Fragmentation
2.2 Precipitation
2.3 Hybridization
2.4 Purification (Prep Station Step)
2.5 Data Collection (Digital Analyzer Step)
2.6 Data Analysis
3 Methods
3.1 Hybridization
3.1.1 Alu1 Restriction Enzyme Digestion
3.1.2 Covaris AFA-Based Fragmentation
3.1.3 Hybridization
3.2 Purification (Prep Station Step)
3.3 Data Collection (Digital Analyzer Step)
3.4 Data Analysis
3.4.1 Raw Data
3.4.2 Normalization
3.4.3 Quality Control
3.4.4 LogRatio Calculation
4 Notes
References
Chapter 19: Cell-Based Methods for the Identification of Myc-Inhibitory Small Molecules
1 Introduction
2 Materials
2.1 Cell Lines
2.2 Lentiviral and Retroviral Vectors
2.3 Other Reagents
2.4 Equipment
3 Methods
3.1 Overview
3.2 Procedure for Screening of Small Molecules for Myc Inhibition
3.3 Filtering for False Positives
3.3.1 Luciferase Inhibition/Quenchers
3.3.2 General Transcription Inhibition
3.4 Cell-Based Assays for Inhibition of Endogenous Myc-Dependent Phenotypes
3.4.1 Proliferation Assay
3.4.2 The Cell Cycle Distribution Assay
4 Notes
References
Index