Zebrafish: Methods and Protocols (Methods in Molecular Biology, 1451)

✍ Scribed by Koichi Kawakami (editor), E. Elizabeth Patton (editor), Michael Orger (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 369
• Category: Library

✦ Synopsis

This second edition details new emerging areas of zebrafish research focusing on genetics and genomics, techniques for developing and analyzing zebrafish disease models, and methods for neuroscience. Zebrafish: Methods and Protocols, Second Edition guides readers through methods for mutagenesis and genome editing in zebrafish, applications of GFP-expressing transgenic fish, techniques for cancer models, imaging of infection and host-pathogen interactions, metabolism and transport of lipids, and the structure and function of neural circuits and their role in generating behavior. 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, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical Zebrafish: Methods and Protocols, Second Edition is a useful complement to the first book for new and experienced zebrafish researcher alike.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Genetics and Genomics
Chapter 1: Chemical Screening in Zebrafish
1 Overview
2 Materials
2.1 Zebrafish Mating
2.2 Raising Larval Zebrafish
2.3 Plating Larvae
2.4 Small Molecule Treatment
3 Methods
3.1 Large-Scale Mating of Zebrafish
3.2 Raising Larval Fish
3.3 Plating Larval Fish in 96-Well Dishes
3.4 Selecting a Chemical Library
3.5 Treating Larvae with Small Molecules
3.6 Phenotypic Readout of Treated Larval Fish
3.7 Data Assessment and Secondary Screening
3.8 Follow-Up Studies: Translation and Mechanism of Action
References
Chapter 2: TALEN-Mediated Mutagenesis and Genome Editing
1 Introduction
2 Materials
2.1 Zebrafish Embryo Genotyping and RFLP Assay
2.2 TALEN Assembling
2.3 In Vitro Transcription
3 Methods
3.1 Designing TALEN with Mojo Hand 2.0 (Fig. 1; See Note 4)
3.2 Genotyping Targeted Genomic Locus
3.3 Design Short Single-Stranded Donor Oligo
3.4 TALEN Assembling with FusX System (3 Days)
3.4.1 Day 1
3.4.2 Day 2
3.4.3 Day 3
3.5 Synthesizing TALEN-Encoding mRNA and Microinjection into One-Cell Zebrafish Embryos
3.6 Examine Somatic TALEN Activity by RFLP Assay or PCR to Detect a Large Deletion
3.7 Screening of Germline Transmission for Stable Mutants
4 Notes
References
Chapter 3: Homology-Independent Integration of Plasmid DNA into the Zebrafish Genome
1 Introduction
1.1 Gene-Trap Screens
1.2 Enhancer-Trap Screens
1.3 Genome-
1.4 Genome Engineering
1.5 Outline of the Procedure
2 Materials
2.1 Zebrafish Strains Used in This Protocol
2.2 Plasmids Used in This Protocol
2.3 Kits Used in This Protocol
2.4 Buffers and Other Reagents
2.5 Equipment
3 Methods
3.1 Design of Locus-­Specific sgRNAs (See Note 1)
3.2 Preparation of Plasmid DNAs for In Vitro Transcription
3.3 Transcription of sgRNAs
3.4 Transcription of Cas9 mRNA
3.5 Efficiency Estimation of sgRNA
3.6 Design of the Donor Vector and Preparation of Donor Plasmid DNA (See Note 21)
3.7 Creation of Transgenic Zebrafish Embryos with Locus-­Specific Integration
3.8 Fluorescent Screening and Confirmation of Locus-­Specific Integration by PCR
3.9 Screening for Germline Transmission in Injected Embryos by Fluorescence and PCR
4 Notes
References
Chapter 4: Detection of Multiple Genome Modifications Induced by the CRISPR/Cas9 System
1 Introduction
2 Materials
2.1 Construction of gRNA Expression Plasmids
2.2 Preparation of gRNA and Cas9 mRNA
2.3 Microinjection
2.4 Preparation of Genomic DNA
2.5 Heteroduplex Mobility Assay (HMA)
2.6 Identification of F0 Founders by Genotyping F1 Embryos
3 Methods
3.1 Construction of gRNA Expression Plasmids
3.2 Preparation of gRNA
3.3 Preparation of Cas9 mRNA
3.4 Microinjection
3.5 Preparation of Genomic DNA
3.6 HMA
3.7 Identification of F0 Founders Using F1 Embryos (See Note 9)
3.8 Determination of the Mutation by Sequencing
4 Notes
References
Chapter 5: Generation of Targeted Genomic Deletions Through CRISPR/Cas System in Zebrafish
1 Introduction
2 Materials
2.1 Reagents and Solutions for Molecular Biology Experiments
2.2 Equipment, Reagents, and Consumable Materials for Zebrafish Husbandry and Microinjection
3 Methods
3.1 Design and Verification of Cas9/gRNA Target Sites
3.2 Preparation of zCas9 mRNA by In Vitro Transcription
3.3 Preparation of gRNAs by In Vitro Transcription
3.4 Generation of Founder Fish by Microinjection of zCas9 mRNA and gRNA Pairs into Zebrafish Embryos
3.5 (Optional) Evaluation of Targeting Efficiency of Individual gRNA in Founder Embryos
3.6 Detection and Evaluation of Chromosomal Deletion Efficiency in Founder Embryos
3.7 Evaluation of Germline Transmission Efficiency of Chromosomal Deletions and Screen for Mutants
4 Notes
References
Chapter 6: Testing of Cis-Regulatory Elements by Targeted Transgene Integration in Zebrafish Using PhiC31 Integrase
1 Introduction
2 Materials
2.1 PhiC31 mRNA Synthesis
2.2 Microinjection and Culturing of the Injected Embryos
3 Methods
3.1 PhiC31 mRNA Synthesis
3.1.1 Vector Template Preparation
3.1.2 In Vitro mRNA Transcription Reaction
3.1.3 Preparation of Donor Plasmid DNA
3.1.4 Preparation of Donor BAC DNA
3.2 Microinjection
3.2.1 Preparation for Microinjection
3.2.2 Microinjections Procedure
3.3 Embryo Culturing, Prescreening, and Screening
4 Notes
References
Chapter 7: Fluorescence-Activated Cell Sorting and Gene Expression Profiling of GFP-Positive Cells from Transgenic Zebrafish Lines
1 Introduction
2 Materials
2.1 Fluorescence-­Activated Cell Sorting of GFP-­Positive Cells from Brain
2.2 RNA Extraction and cDNA Synthesis
2.3 Library Preparation and Sequencing
3 Methods
3.1 Fluorescence-­Activated Cell Sorting of GFP-­Positive Cells from the Brain
3.2 RNA Extraction and cDNA Synthesis
3.2.1 RNA Extraction
3.2.2 cDNA Synthesis
3.2.3 Amplification of cDNA by Long-­Distance PCR
3.3 Library Preparation and Sequence
3.4 Data Analysis
4 Notes
References
Chapter 8: Construction of the Inbred Strain
1 Introduction
2 Materials
2.1 Fish Mating, Collecting Eggs, and Maintenance of Embryos and Fish
2.2 Tetrahymena thermophila Culture
3 Methods
3.1 Inbreeding a Strain
3.2 Breeding the Embryos to the Adults
3.3 Preparation of Tetrahymena thermophila
4 Notes
References
Part II: Disease Models and Mechanism
Chapter 9: Zebrafish as a Model for the Study of Solid Malignancies
1 Introduction
2 Materials
2.1 Gateway Plasmid Constructs and Cloning Reagents [31]
2.2 Synthesis of Transposase mRNA (Refer to [23] for Details)
2.3 Mosaic Transgenesis of Oncogene
2.4 Screening Materials
2.5 Characterizing Embryonic Phenotypes
2.5.1 Phenotype Analysis
2.5.2 FACS Analysis (See Ref. 32 for Details)
2.5.3 Total RNA Isolation
2.5.4 Microscopy and Live Cell Imaging
2.6 Tumor Analysis in Adults
2.6.1 Pathology of Embryos or Resected Tumors
2.6.2 DNA Isolation (of Embryos, Tumors, and Tail Clips)
2.6.3 Total RNA Isolation from Tumor Samples
3 Methods
3.1 Identifying a Gene of Interest (GOI) to Study its Tumorigenic Capacity in a Zebrafish System
3.2 Cloning Your GOI into a Gateway Expression System for Tol2 Transgenesis
3.2.1 Creating 3′ Entry GOI and Control Plasmid Constructs
3.2.2 Creating an Expression System with Your GOI and a Relevant Control
3.3 Injection and Mosaic Expression of Your GOI in Zebrafish Embryos
3.4 Screening Injected Embryos from 24hpf to 5dpf
3.5 Continue Monitoring Larvae and Adults for Cell Masses or Tumors
3.6 Analysis of Embryonic Phenotypes: Overall Survival, Phenotypes, GFP Tissue-Specific Tolerance and Temporal Expression Kinetics, RNA Expression Analysis, and Live Cell Lineage Tracing
3.6.1 Understanding How Oncogenes Affect Early Zebrafish Development
3.6.2 Embryonic Analysis: Overall Survival, Phenotypes, and GFP-GOI Expression Patterns
3.6.3 Embryonic Analysis: Isolation of GFP-GOI-­Positive Cell Populations for Expression Analysis
3.6.4 Embryonic Analysis: Microscopy and Live Cell Imaging
3.7 Analysis of Adult Zebrafish Tumors
3.8 Approach for Analysis of Adult Zebrafish Tumors: Suspected Tumor
3.9 Approach for Analysis of Adult Zebrafish Tumors: Abdominal Tumor
3.10 Approach for Analysis of Adult Zebrafish Tumors: Multiple Tumors
3.11 Histology, Immunohisto-chemistry, and Immuno-fluorescence
3.12 DNA Isolation of Both Tumor and Germline Tissue Samples
3.13 RNA Isolation and Expression Analysis
4 Notes
References
Chapter 10: Melanoma Regression and Recurrence in Zebrafish
1 Introduction
2 Materials
2.1 Zebrafish Strains and Primers
2.2 Zebrafish Maintaining Solutions
2.3 Genotyping Reagents
2.4 Fixation and Histology Solutions
3 Methods
3.1 Generating the Tg(mitfa:BRAF V600E); mitfavc7 line
3.2 Inducing Melanoma Formation, Regression, and Recurrence
3.3 Fixation
3.4 Embedding and Sectioning
3.5 Hematoxylin and Eosin (H&E) Staining
3.6 Immuno-histochemistry
4 Notes
References
Chapter 11: Imaging of Human Cancer Cell Proliferation, Invasion, and Micrometastasis in a Zebrafish Xenogeneic Engraftment Model
1 Introduction
2 Materials
2.1 Tumor Cell Suspension Preparation
2.2 Preparation of Zebrafish Embryos and Microscopy for Phenotype Detection
2.3 Immuno-histochemistry
3 Methods
3.1 Zebrafish Lines and Embryo Preparation
3.2 Tumor Cell Suspension Preparation
3.3 Engraftment of Human Tumor Cells in Zebrafish Embryos
3.4 Verification of Correct Tumor Cell Engraftment in Zebrafish Embryos
3.5 Phenotype Analysis of Tumor Cell Extravasation and Micrometastasis Formation in Zebrafish Larvae
3.6 Phenotype Analysis of Tumor Burden by Automated Imaging
3.7 Immuno
3.8 Testing the Effect of Chemical Compounds on Cancer Proliferation and Micrometastasis Formation In Vivo
3.9 Innate Immunity Interaction with Human Cancer Cells
3.10 Statistics
4 Notes
References
Chapter 12: Modeling Leukemogenesis in the Zebrafish Using Genetic and Xenograft Models
1 Introduction
2 Materials
2.1 Equipment
2.2 Adult Zebrafish and Embryo Handling
2.3 Transgenic Model Development.
2.4 Xenograft Model Development
3 Methods
3.1 Transgenic Leukemia Model
3.1.1 Cloning of Transgenic Vector
3.1.2 Breeding of Fish
3.1.3 Microinjection into Zebrafish Embryos
3.1.4 Genotyping of Injected Zebrafish
3.1.5 Generating F1 Generation and Creating an Active NUP98-HOXA9 Line
3.2 Xenograft: Leukemia Model
3.2.1 Preparing and Storing Primary Leukemia Cells
3.2.2 Day 0: Breeding of Adult Casper Fish
3.2.3 Day 1: Dechorionating
3.2.4 Day 2: Labeling the Leukemia Cell Lines and Primary Samples for Injection
3.2.5 Day 2: Injection
3.2.6 Day 4–8: Drug Screening
3.2.7 Day 6–8: Dissociation Assay for Ex Vivo Quantification of Xenografted Leukemia Cells
3.2.8 Determination of Cell Count and Statistics
4 Notes
References
Chapter 13: Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos
1 Introduction
2 Materials
2.1 Transgenic Lines
2.2 Reagents
2.3 Instru
2.4 Software
2.5 Other Materials
3 Methods
3.1 Choosing Appropriate Transgenic Lines and Preparing for the Assay
3.2 Site-Specific HSC Quantification by Fluorescence Microscopy
3.3 Flow Cytometry
3.4 Cell Culture Assay
4 Notes
References
Chapter 14: Live Imaging of Host–Pathogen Interactions in Zebrafish Larvae
1 Introduction
2 Materials
2.1 Bacterial Preparation
2.2 Injection
2.3 Bacterial Preparation for Infection by Microinjection
2.4 Assaying Bacterial Burden by Microscopy
2.5 Initial Imaging
2.6 Extended Imaging
2.7 Drug Treatment
3 Methods
3.1 Injection
3.2 Caudal Vein Injection
3.3 Hindbrain Ventricle and Otic Vesicle Injection
3.4 Trunk Injection
3.5 Assaying Infection Burden by Microscopy
3.6 Initial Imaging
3.7 Extended Time-Lapse Imaging of Infection
3.8 Drug Treatment of Infected Zebrafish
4 Notes
References
Chapter 15: Methodologies for Inducing Cardiac Injury and Assaying Regeneration in Adult Zebrafish
1 Introduction
2 Materials
3 Methods
3.1 Zebrafish Heart Surgery
3.2 Zebrafish CM Ablation
3.3 Histological Analysis for Cardiac Muscle Ablation and Regeneration
3.4 Assays for CM Proliferation During Heart Regeneration
4 Notes
References
Chapter 16: Studying Lipid Metabolism and Transport During Zebrafish Development
1 Introduction
2 Materials
2.1 Embryo/Larvae Collection and Cleaning
2.2 Storage of Chicken Egg Yolk
2.3 Labeling Oil with Fluorescently Tagged Fatty Acids
2.4 Labeling Oil with Radiolabeled Fatty Acids
2.5 Injecting Labeled Oil Droplet into Yolk of Embryos or Larvae
2.6 Preparing BODIPY® Fatty Acid Analogs
2.7 Preparing Chicken egg Yolk Emulsion and Labeling Liposomes with BODIPY® Fatty Acid Analogs
2.8 Feeding Fluorescently Labeled Liposome Solution to Larvae 6 dpf and Older
2.9 Long-Term Live Imaging by Upright Microscopy Using an Immersion Objective
2.10 Long-Term Live Imaging by Inverted Microscopy Using a Standard Objective
2.11 Short-Term Live Imaging by Upright or Inverted Microscopy Using a Standard Objective
2.12 Isolation of Total Lipids Following the Bligh and Dyer Method [23]
2.13 Running TLC Plate Using a Two-­Solvent System
2.14 Analyzing TLC of Fluorescent Lipids
2.15 Analyzing TLC of Radiolabeled Lipids
2.16 Quantifying Ingestion of BODIPY®-Labeled Liposomes
3 Methods
3.1 Embryo/Larvae Collection and Cleaning
3.2 Storage of Chicken Egg Yolk
3.3 Labeling Oil with Fluorescently Tagged Fatty Acids
3.4 Labeling Oil with Radiolabeled Fatty Acids
3.5 Injecting Labeled Oil Droplet into Yolk of Embryos or Larvae
3.6 Preparing BODIPY® Fatty Acid Analogs
3.7 Preparing Chicken Egg Yolk Emulsion and Labeling Liposomes with BODIPY® Fatty Acid Analogs
3.8 Feeding Fluorescently Labeled Liposome Solution to Larvae 6 dpf and Older (Fig. 2a)
3.9 Long-Term Live Imaging by Upright Microscopy Using an Immersion Objective (Fig. 2c)
3.10 Long-Term Live Imaging by Inverted Microscopy Using a Standard Objective (Fig. 2d)
3.11 Short-Term Live Imaging by Upright or Inverted Microscopy Using a Standard Objective (Fig. 2b, e)
3.12 Isolation of Total Lipids Following the Bligh and Dyer Method [23]
3.13 Running TLC Plate Using a Two-Solvent System (Fig. 2f)
3.14 Analyzing TLC of Fluorescent Lipids
3.15 Analyzing TLC of Radiolabeled Lipids
3.16 Quantifying Ingestion of BODIPY®-Labeled Liposomes (Fig. 2g)
4 Notes
References
Part III: Neuroscience
Chapter 17: Targeted Electroporation in Embryonic, Larval, and Adult Zebrafish
1 Introduction
2 Materials
2.1 Instrumentation
2.2 Small Equipment
2.3 Reagents and Solutions
3 Methods
3.1 External-
3.2 Internal-
3.3 Pipette-Electrode Electroporation (PEP)
4 Notes
References
Chapter 18: Studying Axonal Regeneration by Laser Microsurgery and High-Resolution Videomicroscopy
1 Introduction
2 Materials
2.1 Embryo Culture and Injection
2.2 Embryo Selection and Mounting
2.3 Laser Axotomy and Analysis of Neuron Response
3 Methods
3.1 Marking Single Lateralis Neurons in Zebrafish
3.2 Laser Axotomy and Imaging
3.3 Quantification of Axonal Regeneration
4 Notes
5 Ethical Considerations
References
Chapter 19: In Vivo Whole-Cell Patch-Clamp Recording in the Zebrafish Brain
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment for Preparation and Dissection
2.3 Equipment for Electrophysiology (Fig. 2)
3 Methods
3.1 Preparation and Dissection
3.2 Whole-Cell Patch-Clamp Recording
4 Notes
References
Chapter 20: Quantifying Aggressive Behavior in Zebrafish
1 Introduction
2 Materials
3 Methods
3.1 Animal Housing
3.2 Individual Tagging
3.2.1 Fin Clips
3.2.2 Color Tagging with Nylon Monofilament
3.2.3 Recovery from Anesthesia
3.3 Behavioral Recording
3.3.1 Staged Fights
3.3.2 Mirror-Elicited Fights
3.3.3 Quantitative Behavioral Analysis
3.3.4 Typical Results
4 Notes
References
Chapter 21: Correlating Whole Brain Neural Activity with Behavior in Head-Fixed Larval Zebrafish
1 Introduction
2 Materials
2.1 Embedding Larvae
2.2 Imaging Rig
2.3 Data Analysis
3 Methods
3.1 Embedding Larvae
3.2 Two-Photon Microscope
3.3 Data Analysis
3.3.1 Elimination of Motion Artifacts
3.3.2 Unbiased Identification of Functionally Active Units
3.3.3 Analysis of Behavior-Activity Correlations
4 Notes
References
Chapter 22: A Practical Guide to Light Sheet Microscopy
1 Introduction
2 Materials
2.1 Detection Optics: Suggested Parts List
2.2 Excitation Optics: Suggested Parts List
2.3 Sample Chamber
2.4 Control Equipment
2.5 Support Equipment
3 Methods
3.1 Assembly of the Sample Stage
3.2 Assembly of the Detection Arm
3.3 Assembly of the Excitation Arm
3.4 Alignment of Excitation Laser
3.5 Fine Alignment and Light Sheet Generation
3.6 Operation of the Light Sheet Microscope
3.7 Alternative Approaches
4 Notes
References
Chapter 23: Calcium Imaging of Neuronal Activity in Free-Swimming Larval Zebrafish
1 Introduction
2 Materials
2.1 Preparation of Zebrafish Larvae
2.2 Preparation of Paramecia
2.3 Recording Chamber
2.4 Calcium Imaging Equipment
3 Methods
3.1 Preparation of the Larvae
3.2 Paramecium Culture
3.3 Preparation of Paramecia for the Use of Prey Capture Behavioral Recording
3.4 Preparation of a Recording Chamber
3.5 Calcium Imaging
4 Notes
References
Chapter 24: Fiber Optic-Based Photostimulation of Larval Zebrafish
1 Introduction
2 Materials
2.1 Equipment for Fiber Optic Photostimulation
2.2 Reagents and Equipment for Embedding Larvae
3 Methods
3.1 Choice of the Illumination Intensity
3.2 Preparation of the Blunt End Optic Fiber
3.3 Mounting a Larval Zebrafish in a Petri Dish
3.4 Positioning the Optic Fiber
3.5 Photostimulation Control
4 Notes
References
Chapter 25: Genetic Ablation, Sensitization, and Isolation of Neurons Using Nitroreductase and Tetrodotoxin-Insensitive Channels
1 Introduction
2 Materials
2.1 Embryo Rearing
2.2 Ablation and Isolation Reagents
2.3 Fish Stocks
3 Methods
3.1 Raising Larvae for Behavior Testing
3.2 Ablation Using Nitroreductase and Metronidazole
3.3 Sensitization of Neurons by Targeted Expression of Nav1.5
3.4 Isolation of Neurons from Circuit Activity with Nav1.5 and Tetrodotoxin
4 Notes
References
Index