Zebrafish: Methods and Protocols (Methods in Molecular Biology, 2707)

✍ Scribed by James F. Amatruda (editor), Corinne Houart (editor), Koichi Kawakami (editor), Kenneth D. Poss (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 361
• Category: Library

✦ Synopsis

This fully updated edition introduces new tools, models, and analytic insights that position the zebrafish even more strongly as an engine of discovery for developmental and disease biology. Beginning with a section exploring detailed methods for use of zebrafish to model a variety of human diseases, the book continues by illuminating the key ongoing role of the fish model in studies of the vertebrate nervous system, tools and approaches using zebrafish to study stem cell and regenerative biology, as well as techniques in genetics and genomics. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, as well as tips on troubleshooting and avoiding known pitfalls.
Authoritative and up-to-date, Zebrafish: Methods and Protocols, Third Edition serves as an invaluable guide to propel advances in developmental biology, disease modeling, and regeneration research using zebrafish and medaka as model systems.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Disease Models
Chapter 1: Delivering Traumatic Brain Injury to Larval Zebrafish
1 Introduction
2 Materials
2.1 Inducing TBI
2.2 Setup of Pressure Transducer for Measuring Pressure Within the Syringe
3 Methods
3.1 Inducing TBI
3.2 Setup of Pressure Transducer for Pressure Measurement Within the Syringe
3.3 Pressure Measurement within the Syringe
4 Notes
References
Chapter 2: Functional Genomics of Novel Rhabdomyosarcoma Fusion-Oncogenes Using Zebrafish
1 Introduction
2 Materials
2.1 Zebrafish Lines
2.2 Cloning Materials
2.2.1 Plasmids
2.2.2 Supplies
2.3 Injection Materials
2.3.1 Injection Apparatus
2.3.2 Injection Components
2.3.3 Tol2 mRNA Synthesis for DNA Injections
2.3.4 Injection Mixes and Preparation
2.4 Zebrafish Screening Material
2.4.1 Microscope
2.4.2 Embryo Preparation
2.5 Tumor Identification and Collection
2.5.1 Tumor Identification
2.5.2 Fish Euthanasia and Collection of Tumor Samples
2.5.3 Liquid Nitrogen Collection
2.5.4 Histology Collection
2.6 Histological Assessment
2.6.1 Hematoxylin and Eosin Stain
2.6.2 RT-PCR for myoD, myoG, Desma
2.6.3 Primer Sequences for Gene Targets
2.6.4 Immunohistochemistry
2.7 RNA Isolation Materials
2.7.1 Sample/RNA Preparation
2.7.2 RNA Library Preparation
2.8 Statistical Analysis
3 Methods
3.1 Choosing a Rhabdomyosarcoma Fusion-Oncogene to Study
3.2 Cloning Your Fusion Protein into a Gateway Expression Plasmid for Tol2 Transgenesis
3.2.1 Create 3′ Entry Fusion and Control Plasmids
3.2.2 Create Expression Constructs Using Either Gene Fusion 3′ Entry or Control Plasmid
3.3 Injecting Expression Construct
3.4 Screening for Tumors
3.5 Ending the Experiment
3.6 Downstream Processing (See Note 3)
3.6.1 RNA-Seq
3.6.2 RT-PCR
3.6.3 Histology and Immunohistochemistry
3.7 Verify that Zebrafish Tumors Recapitulate the Human Disease
4 Notes
References
Chapter 3: Methods to Study Liver Disease Using Zebrafish Larvae
1 Introduction
2 Materials
2.1 Generating, Raising, Imaging, and Manipulating Zebrafish Larvae
2.2 Apoptosis Using TUNEL Assay
2.3 Senescence Beta-Galactosidase Staining
2.4 Cell Proliferation Using EdU Incorporation
2.5 Assessing Hepatocyte Organelle Dynamics in Liver Disease Models by Live Imaging
2.6 Assessing Fibrosis
2.7 Equipment
2.8 Software
3 Methods
3.1 Liver Size Measurements
3.2 Gene Expression Analysis in Microdissected Zebrafish Larval Livers
3.3 Assessing Tumor Suppression
3.3.1 Apoptosis Using TUNEL Assay
3.3.2 Senescence-Associated (SA) β-Galactosidase Staining
3.4 Liver Cell Division Using EdU Incorporation
3.5 Assessing Hepatocyte Organelle Dynamics in Liver Disease Models by Live Imaging
3.6 Assessing Fibrosis
4 Notes
References
Chapter 4: Developmental Toxicity Assessment Using Zebrafish-Based High-Throughput Screening
1 Introduction
2 Materials
3 Methods
3.1 Fish Husbandry and Feeding
3.2 Embryo Spawning
3.3 Embryo Sorting
3.4 Dechorionation
3.5 Loading into 96-Well Plates
3.6 Waterborne Exposure of Chemicals
3.7 24 h Mortality and Developmental Progression Assessment
3.8 Embryonic Photomotor Response (EPR)
3.9 Larval Photomotor Response (LPR) and Larval Startle Response (LSR)
3.10 Mortality and Morphology Assessments at 120 hpf
3.11 Statistics
3.12 Quality Control
4 Notes
References
Chapter 5: Cancer Modeling by Transgene Electroporation in Adult Zebrafish (TEAZ)
1 Introduction
2 Materials
2.1 Zebrafish Lines
2.2 Instrumentation
2.3 Plasmid Preparation Materials
2.4 Electroporation
2.5 Microscopy
2.6 CRISPR-SEQ
3 Methods
3.1 Plasmid Cloning to Overexpress or Knockout Genes
3.1.1 Overexpression Plasmids: Construct Plasmids Using the Tol2Kit by Gateway Cloning
3.1.2 CRISPR Plasmids: Generate Cell-Type Specific Cas9 Plasmid and gRNA Plasmids
3.2 Electroporation
3.2.1 Preparation of Injection Mix
3.2.2 Prepare Injection Plate
3.2.3 Set Up Workspace
3.2.4 Prepare Injection Needle
3.2.5 Microinjection of DNA into Fish
3.2.6 Electroporation of Fish
3.3 CRISPR-Sequencing
3.3.1 Primer Design
3.3.2 Sample Preparation
3.3.3 PCR to Generate CRISPR Seq Amplicons
3.4 Microscopy
4 Notes
References
Chapter 6: Lineage Tracing of Bone Cells in the Regenerating Fin and During Repair of Bone Lesions
1 Introduction
2 Materials
2.1 Medaka Lines
2.2 Reagents
2.3 Equipment and Consumables
3 Methods
3.1 Lineage Tracing of col10a1 Cells During Bone Repair in the Medaka Vertebral Column
3.1.1 Preparation of Transgenic Embryos for Lineage Tracing
3.1.2 Induction of Cre/LoxP Recombination by 4-Hydroxytamoxifen: Labeling of col10a1-Positive Osteoblast Progenitors
3.1.3 Induction of Osteoporosis-Like Bone Lesions in the Medaka Larval Vertebral Column
3.1.4 In Vivo Tracking of Labeled Cells During Repair of Bone Lesions
3.2 Lineage Tracing of col10a1 Cells During Medaka Adult Fin Regeneration
3.2.1 Preparation of Transgenic Embryos for Lineage Tracing
3.2.2 Induction of Cre/LoxP Recombination-Labeling of col10a1 Cells in Adult Medaka
3.2.3 Fin Amputation and In Vivo Tracking of Labeled Cells in the Regenerating Fin
4 Notes
References
Part II: Neuroscience
Chapter 7: Optimized Primary Culture of Neuronal Populations for Subcellular Omics Applications
1 Introduction
2 Materials
2.1 Embryo Dissociation
2.2 Primary Cell Culture
2.3 Primary Culture Using Transwell Inserts
2.4 Tissue Isolation from Transwell Inserts and RNA Extraction
3 Methods
3.1 Embryo Dissociation
3.2 Primary Cell Culture
3.3 Primary Culture Using Transwell Inserts
3.4 Neurite Isolation and RNA Extraction
4 Notes
References
Chapter 8: Holographic Optogenetic Activation of Neurons Eliciting Locomotion in Head-Embedded Larval Zebrafish
1 Introduction
2 Materials
2.1 Preparations of Larval Zebrafish
2.2 Light Patterning Protocol
2.3 Behavioral Monitoring
3 Methods
3.1 Expression of Opsins and Selection of Transgenic Larvae
3.2 Preparation of the Larvae Before Experiment
3.3 Calibration of the Holographic Illumination
3.3.1 Spatial Calibration of the Holographic Beam
3.3.2 Power Calibration of the Holographic Illumination
3.4 Monitoring the Behavioral Output
3.5 The Optogenetic Experiment
3.5.1 Choosing the Parameters of Stimulation
3.6 Photostimulation Control
3.6.1 Controls in Non-opsin-Expressing Larvae
3.6.2 Out-of-Target Optogenetic Stimulation in Larvae Expressing the Opsin
3.6.3 Ablation Control to Inspect the Contribution of the Targeted Neuron(s)
4 Notes
References
Chapter 9: Brain Imaging and Registration in Larval Zebrafish
1 Introduction
2 Materials
2.1 Reagents and Equipment for Mounting and Imaging Embryos
2.2 Brain Registration
3 Methods
3.1 Select a Registration Channel
3.2 Raise Larvae
3.3 Label Brain Tissue with a Fluorescent Marker
3.4 Mount Larvae for Imaging
3.5 Confocal Imaging
3.6 Post-imaging Processing
3.7 Brain Registration
4 Notes
References
Chapter 10: Simultaneous Behavioral and Neuronal Imaging by Tracking Microscopy
1 Introduction
2 Materials
2.1 Materials for Chamber Assembly
2.2 Material for Thin Fluorescent Sheet
3 Methods
3.1 Chamber Assembly
3.2 Collection of Calibration Data with Thin Fluorescent Sheet
3.3 Collection of Reference Brain Stack for Online and Offline Registration
3.4 Data Acquisition During Experiment
3.5 DIFF Solve Post-imaging
3.6 Registration Post-imaging
4 Notes
References
Chapter 11: Genetic Identification of Neural Circuits Essential for Active Avoidance Fear Conditioning in Adult Zebrafish
1 Introduction
2 Materials
2.1 Identification of Brain-Specific Transgenic Zebrafish Lines
2.2 Active Avoidance Fear Conditioning
2.3 Analysis of Neural Circuits Mediating Active Avoidance Fear Conditioning
3 Methods
3.1 Identification of Brain-Specific Transgenic Zebrafish Lines
3.2 Active Avoidance Fear Conditioning
3.2.1 Setting Up the Behavior Arena
3.2.2 Habituation
3.2.3 Training Protocol
3.2.4 Data Analysis
3.2.5 Inhibiting Neuronal Activity in Adult Zebrafish Brain
3.3 Analysis of Neural Circuits Mediating Avoidance Fear Learning
4 Notes
References
Part III: Regeneration
Chapter 12: Quantitative Live Imaging of Zebrafish Scale Regeneration: From Adult Fish to Signaling Patterns and Tissue Flows
1 Introduction
2 Materials
2.1 Scale Injury
2.2 Scale Regeneration Imaging
2.3 Laser-Mediated Photoconversion
2.4 Scale Image Processing
2.5 Scale Image Quantification: Nuclear Segmentation and Reporter Signal Quantification
2.6 Scale Image Quantification: Tissue Flows
3 Methods
3.1 Scale Injury
3.2 Scale Regeneration Imaging
3.3 Laser-Mediated Photoconversion
3.4 Scale Image Processing
3.5 Image Quantification: Nuclei Segmentation and Reporter Signal Quantification
3.6 Image Quantification: Tissue Flows
4 Notes
References
Chapter 13: Generation of Conditional Knockout Zebrafish Using an Invertible Gene-Trap Cassette
1 Introduction
2 Materials
2.1 Solutions
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Identification of Insertion Sites
3.2 Generation and Efficiency Test of TALENs
3.3 In Vivo Homologous Recombination
3.4 Founder Fish Identification
3.5 Induction of cKO and Assessment of Inactivation Efficiency
4 Notes
References
Chapter 14: Spinal Cord Injury and Assays for Regeneration
1 Introduction
2 Materials
2.1 Spinal Cord Injury
2.2 Swim Endurance Assay
2.3 Histology
3 Methods
3.1 Spinal Cord Injury
3.2 Swim Tunnel Preparation and Calibration
3.3 Assessment of Swim Endurance
3.4 Axon Tracing with Biocytin
3.5 Histology
3.6 Imaging and Quantification of Glial and Axonal Bridging
4 Notes
References
Chapter 15: Selective Cell Ablation Using an Improved Prodrug-Converting Nitroreductase
1 Introduction
2 Materials
2.1 Reagents
2.2 Solutions
3 Methods
3.1 Cell Ablation Protocol
3.2 Detecting Cell Loss
4 Notes
References
Chapter 16: Section Immunostaining for Protein Expression and Cell Proliferation Studies of Regenerating Fins
1 Introduction
2 Materials
2.1 Equipment and Supplies
2.2 Required Buffers and Reagents
3 Methods
3.1 Intraperitoneal Injection of Adult Zebrafish with 5-Ethynyl-2′-Deoxyuridine (EdU)
3.2 Tissue Collection and Fixation
3.3 Preparation of Paraffin Sections
3.4 Preparation of Frozen Sections
3.5 Dewaxing and Hydration of Paraffin Sections
3.6 Hydration of Frozen Sections
3.7 Antigen Retrieval
3.8 Blocking Nonspecific Antibody Binding
3.9 EdU Detection
3.10 Immunostaining
3.11 Imaging and Data Interpretation
4 Notes
References
Part IV: Genetics and Genomics
Chapter 17: In Vivo Optogenetic Phase Transition of an Intrinsically Disordered Protein
1 Introduction
2 Materials
2.1 Expression Construct for Optogenetic TDP-43
2.2 Solutions and Reagents
2.3 Equipment for Optogenetics
2.4 Microscopy
3 Methods
3.1 BAC-Mediated Expression of opTDP-43 in the Spinal Motor Neurons
3.2 Light-Induced Phase Transition of opTDP-43h
4 Notes
References
Chapter 18: Colorimetric Barcoding to Track, Isolate, and Analyze Hematopoietic Stem Cell Clones
1 Introduction
2 Materials
2.1 Genotyping of Tg(ubi:Zebrabow-M)
2.2 Zebrafish
2.3 creERT2-Mediated Recombination
2.4 Cardiac Bleed and Kidney Marrow Collection
2.5 Analysis of the Recombined Kidney Marrow
3 Methods
3.1 Genotyping of Adult Tg(ubi:Zebrabow-M)
3.2 Genotyping of Tg(drl:creERT2)
3.3 Zebrabow Fish Breeding and Color Induction
3.4 Determine Hematopoietic Clone Number and Clone Size
4 Notes
References
Chapter 19: Mutation Knock-in Methods Using Single-Stranded DNA and Gene Editing Tools in Zebrafish
1 Introduction
2 Materials
2.1 General Molecular Biology Reagents
2.2 Growing and Handling ZebrafishZebrafish
3 Methods
3.1 Sequencing of the Target Region in the ZebrafishZebrafish Strain(s) Used for Experiments
3.2 Design of ssODN for Introducing Short Indel Modifications by CRISPR/Cas9CRISPR/Cas9
3.3 Design of ssODN for Introducing Amino Acid Changes by CRISPR/Cas9CRISPR/Cas9
3.3.1 Manual Design of Oligos for Amino Acid Changes
3.3.2 Design of Amino Acid Change Point Mutations Using CRISPR Knock-in Designer
3.4 Design and Preparation of lssDNA Donor Template Oligos
3.4.1 Synthetic lssDNA
3.4.2 Reverse Transcription-Based Synthesis of lssDNA (Fig. 6c)
3.4.3 Exonuclease-Based Production of lssDNA (Fig. 6d)
3.5 Design and Preparation of Chimeric RNA:DNA Donorguide Molecules
3.6 Reagent Preparation, Microinjection of ZebrafishZebrafish Eggs and Sample Collection
3.7 Detection of Precise and Imprecise Knock-in Edits
3.7.1 Allele-Specific PCRPolymerase chain reaction (PCR)
3.7.2 Preparation of Samples for Deep Sequencing
4 Notes
References
Chapter 20: Generation of Transgenic Fish Harboring CRISPR/Cas9-Mediated Somatic Mutations Via a tRNA-Based Multiplex sgRNA Ex...
1 Introduction
2 Materials
2.1 Microinjection of sgRNAs, Cas9 mRNA, and Plasmids
2.2 Heteroduplex Mobility Assay (HMA)
2.2.1 Heteroduplex Mobility Assay with Polyacrylamide Gel Electrophoresis (PAGE)
2.2.2 Heteroduplex Mobility Assay with MultiNA (SHIMADZU)
2.3 Construction of Plasmids Containing tRNA-sgRNA Tandem Repeats
3 Methods
3.1 Design and Preparation of sgRNAs
3.2 Microinjection of Cas9 mRNA and sgRNAs
3.3 Evaluation of the Efficiency of sgRNAs by the Heteroduplex Mobility Assay
3.4 Construction of Plasmids Containing tRNA-sgRNA Tandem Repeats
3.5 Generation of Transgenic Fish and Detection of Somatic Mutations
4 Notes
References
Chapter 21: Scalable CRISPR Screens in Zebrafish Using MIC-Drop
1 Introduction
2 Materials
2.1 Zebrafish Husbandry and Maintenance
2.2 MIC-Drop Library Generation
2.3 MIC-Drop Library Injection, Phenotyping, and Validation
3 Methods
3.1 sgRNA Design and Synthesis
3.2 Barcode Generation
3.3 Droplet Generation
3.4 Droplet Microinjection
3.5 Phenotyping and Barcode Retrieval
3.6 Identification of Candidate ``Hits´´ and Validation
4 Notes
References
Chapter 22: The Goldfish Genome and Its Utility for Understanding Gene Regulation and Vertebrate Body Morphology
1 Introduction
2 Materials
2.1 Fish
2.2 Anesthetization of Goldfish
2.3 Genomic DNA Purification from Goldfish Tissues
2.4 NGS Sequencers
2.5 Voltage Clamp Recording
2.6 Identification of the 13-kbp Insertion in the Telescope-Eye Goldfish Genome
2.7 CRISPR/Cas9-Mediated Gene Disruption of lrp6 in Zebrafish
3 Methods
3.1 Introduction to Comparative Genomics
3.2 Comparative Genomics Using the Goldfish and Zebrafish Genomes
3.3 Comparisons Within the Goldfish Genome
3.4 Identifying Loci Associated with Goldfish Phenotypes
3.4.1 Genomic DNA Purification
3.4.2 GWAS Analysis 1: The Long-Tail Phenotype
3.4.3 GWAS Analysis 2: The Twin-Tail Phenotype
3.4.4 GWAS Analysis 3: The Telescope-Eye Phenotype
3.4.5 GWAS Analysis 4: The Dorsal Fin Loss Phenotype
3.4.6 Strain-Specific Variant (SSV) Analysis: Albinism
4 Conclusions
References
Index