C. elegans: Methods and Applications

Preface
Contents
Contributors
Chapter 1: Starting a C. elegans Research Laboratory: Practical Advice
	1 Introduction
		1.1 Who we Are Writing this for
		1.2 We Hope to Provide
		1.3 We Are Not Providing
	2 Joining the Community
		2.1 Spend Time in an Existing C. elegans Lab
		2.2 Community Resources and Organizations You Should Know About
		2.3 Meetings and Conferences
		2.4 Communication
		2.5 Connecting with your Local C. elegans Community
	3 Setting up your C. elegans Lab
		3.1 Organizing
		3.2 Equipment
		3.3 C. elegans Husbandry
		3.4 Organizing
	4 C. elegans Nomenclature, Data Management, Dissemination of Results and Reagents
		4.1 Nomenclature
		4.2 Distribution of C. elegans Strains and Reagents
		4.3 Data Management for your Lab
		4.4 Publication of Results
	5 What I Wish Knew When I Started: Advice to New Colleagues
		5.1 Onboarding
		5.2 Develop Communication Policies
		5.3 Turn off Notifications
		5.4 Schedule Working Meetings
		5.5 Supporting Students and Mentees
		5.6 Stay in Contact with your Mentors
		5.7 Inclusive Environment
	6 Distributables for New Lab Members: Three Sample Handouts
		6.1 Sample Handout 1
			6.1.1 Accumulated Wisdom: Guidelines for Successful C. elegans Studies
			6.1.2 Lab Rules of the Road
		6.2 Sample Handout 2
			6.2.1 Welcome to Working at the Bench in our Lab
				Development of your Project
				Teach Others What You Have Learned
				Laboratory Maintenance: Laboratory Chores and Duties
				Research Meetings
				Data Management
				From Data Collection to Manuscripts
				Authorship and Publications
				Conference Attendance and Travel
				Lab Communication and Time Spent in Lab
				Mentorship, Assessing Your Progress, Recommendation Letters and Additional Expectations
		6.3 Sample Handout 3
			6.3.1 Expectations for all Laboratory Undergraduates
			6.3.2 Before you Join the Laboratory
				After You Have a Research Project and Join the Laboratory
				Undertaking Summer Research
Chapter 2: The Basics of Setting up Successful Teaching Labs and Short-Term Projects with C. elegans
	1 Introduction
	2 Materials
		2.1 C. elegans Maintenance
		2.2 Chemotaxis Assays
	3 Methods
		3.1 Maintaining C. elegans: ``Picking Worms´´ to Transfer C. elegans
		3.2 Maintaining C. elegans: ``Chunking´´ to Transfer C. elegans
		3.3 Maintaining C. elegans: Making and Thawing C. elegans Frozen Stocks
		3.4 Maintaining C. elegans: Removing Contamination by Bleaching C. elegans
		3.5 Instructor Preparation for Chemotaxis Teaching Lab
		3.6 Making the Classroom Laboratory an Inclusive Environment for all Students
		3.7 Example of Teaching Lab Module for Students
			3.7.1 Introduction
			3.7.2 Chemotaxis Materials
			3.7.3 Chemotaxis Methods
		3.8 Discussion of Chemotaxis Data
		3.9 Chemotaxis Extension: Testing Novel Odorants in C. elegans Chemotaxis
	4 Notes
	References
Chapter 3: Cryopreservation of C. elegans and Other Nematodes with Dimethyl Sulfoxide and Trehalose
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 4: Genetic Methods for Cellular Manipulation in C. elegans
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Buffers
		2.3 Other
	3 Methods
		3.1 Preparing Injection Pads (See Note 5)
		3.2 Preparing Microinjection Needles
		3.3 Preparing DNA for Injection
		3.4 Loading the Needle with  DNA
		3.5 Mounting the Needle on the Microscope and Breaking the Needle
		3.6 Mounting the Worm on the Injection  Pad
		3.7 Injection
		3.8 Recovery of the  Worm
		3.9 Selection for Transgenic Worms
	4 Notes
	References
Chapter 5: Observing and Quantifying Fluorescent Reporters
	1 Introduction
		1.1 Fluorescent Molecules and Microscopy
		1.2 Fluorescent Proteins in Biology
	2 Materials
		2.1 Fluorescent Reporters Used in C. elegans
			2.1.1 Static Fluorescent Reporters
			2.1.2 Calcium
			2.1.3 Voltage
			2.1.4 Physiological Reporters
		2.2 Transgenic Worms
		2.3 Microscope Equipped with Fluorescence Components
	3 Methods
		3.1 Mounting and Immobilization
		3.2 Acquiring Images
		3.3 Image Analysis and Measurement
	4 Notes
	References
Chapter 6: Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Ac...
	1 Introduction
	2 Materials
		2.1 Optogenetic Activation: Depolarizing Membrane Potential
			2.1.1 Channelrhodopsin-2
			2.1.2 ChR2 Variants with Distinct Functional Properties for Specific Applications
		2.2 Optogenetic Inhibition: Hyperpolarizing Membrane Potential
			2.2.1 Halorhodopsin
			2.2.2 Archaerhodopsin and  Mac
			2.2.3 Voltage Imaging Tools
			2.2.4 Anion Channelrhodopsins (ACRs)
		2.3 Guanylyl Cyclase Rhodopsin (CyclOp)
	3 Methods
		3.1 Specific Application of Optogenetics to Investigate Synaptic Function
			3.1.1 Expression of Optogenetic Proteins
			3.1.2 Worm Rearing
			3.1.3 Illumination
			3.1.4 Video Analysis
		3.2 Combining Optogenetic Actuation with Ca2+ Imaging
		3.3 Voltage Imaging Using Rhodopsins
		3.4 Cyclase Rhodopsins and Two-Component Optogenetics
	4 Notes
	References
Chapter 7: Optogenetic Perturbation of Individual C. elegans Pharyngeal Neurons While Monitoring Feeding Behavior
	1 Introduction
	2 Materials
	3 Methods
		3.1 Building the  Rig
		3.2 Creating Worm Strains
		3.3 Preparing Agarose  Pads
		3.4 Performing the Experiments
		3.5 Analyzing the  Data
	4 Notes
	References
Chapter 8: Antibody Staining for Nematodes with Heat-induced Antigen Retrieval (HIAR)
	1 Introduction
	2 Materials
		2.1 ``Improved Finney-Ruvkun´´ Method Fixation Recipes (See Note 1)
		2.2 Cultivation Reagents
		2.3 Equipment
	3 Methods
	4 Notes
	References
Chapter 9: ExCel: Super-Resolution Imaging of C. elegans with Expansion Microscopy
	1 Introduction
	2 Materials
		2.1 Common for all ExCel Protocols
			2.1.1 Fixation
			2.1.2 RNA and Protein Anchoring
			2.1.3 Gelation and Expansion
			2.1.4 General Purpose Buffer
			2.1.5 Antibody Staining
		2.2 Standard Expansion of C. elegans
			2.2.1 ExFISH-HCR (for RNA Readout)
		2.3 Epitope-Preserving Expansion of C. elegans
		2.4 Iterative Expansion of C. elegans (iExCel)
	3 Methods
		3.1 Standard Expansion of C. elegans (ExCel)
			3.1.1 Overview
			3.1.2 Fixation and Cuticle Reduction
			3.1.3 Sample Allocation
			3.1.4 RNA Anchoring (Optional, for RNA Readout)
			3.1.5 Protein Anchoring (for Fluorescent Protein Readout)
			3.1.6 Selecting a Method to Track Hydrogel Orientation
			3.1.7 Gelation
			3.1.8 Digestion
			3.1.9 ExFISH-HCR (for RNA Readout): Re-Embedding
			3.1.10 ExFISH-HCR (for RNA Readout): Probe Hybridization and HCR Amplification
			3.1.11 Antibody Staining (for Fluorescent Protein Readout)
			3.1.12 NHS-Ester Staining (Optional, for Morphology Readout)
			3.1.13 DAPI Staining (Optional, for DNA Readout)
			3.1.14 Preliminary Imaging at a Partially Expanded State (Only for Samples that Have Not Been Re-Embedded)
			3.1.15 Final Imaging at Fully Expanded State
			3.1.16 Agar Immobilization (Optional, for Vibration-Free Imaging)
			3.1.17 Example of Final Images
		3.2 Epitope-Preserving Expansion of C. elegans
			3.2.1 Overview
			3.2.2 Gelation
			3.2.3 Collagenase VII-Mediated Cuticle Digestion
			3.2.4 Protein Denaturation
			3.2.5 Antibody Staining
			3.2.6 Preliminary Imaging to Check Antibody Staining
			3.2.7 Antibody Anchoring
			3.2.8 Re-Embedding into an Expandable Second  Gel
			3.2.9 Proteinase K Digestion
			3.2.10 Cleave DATD-Crosslinked Gels #1
			3.2.11 NHS-Ester Staining (Optional, for Morphology Readout)
			3.2.12 Expansion and Imaging
			3.2.13 Example of Final Images
		3.3 Iterative Expansion of C. elegans (iExCel)
			3.3.1 Overview
			3.3.2 Pre-iterative ExCel Notes and Protocols
			3.3.3 Protocol for Synthesizing DNA-Conjugated Secondary Antibody for iExCel
			3.3.4 Antibody Staining (for Fluorescent Protein Readout)
			3.3.5 Expansion and Re-Embedding into Non-Expanding Gel #2
			3.3.6 Linker Hybridization (to Amplify and Transfer Signals from Gel#1 to Gel#3)
			3.3.7 Re-Embedding into Expanding Gel #3
			3.3.8 Cleave DATD-Crosslinked Gels #1 and #2
			3.3.9 LNA Hybridization (to Attach Fluorophores to the Stained Locations for Final Readout)
			3.3.10 Expansion and Imaging
			3.3.11 Example of Final Images
	4 Notes
	References
Chapter 10: A Fusion PCR Method for Expressing Genetic Tools in C. elegans
	1 Introduction
	2 Materials
		2.1 Promoter Cloning
		2.2 Cloning Reporter  Gene
		2.3 Fusion PCR
	3 Methods
		3.1 Amplifying the Promoter from Genomic  DNA
		3.2 Cloning and Amplifying the Gene of Interest
		3.3 Fusion PCR of the Promoter and  Gene
	4 Notes
	References
Chapter 11: Approaches for CRISPR/Cas9 Genome Editing in C. elegans
	1 Introduction
	2 Materials
		2.1 sgRNA, Template DNA Generation, and Cloning of Homology Arm Vectors
		2.2 Microinjection of C. elegans for CRISPR/Cas9 Genome Editing and Screening Animals
	3 Methods
		3.1 Considerations before Starting Editing Experiments
		3.2 Selecting Guide RNAs for Performing CRISPR Editing in C. elegans
		3.3 Selecting Homology Arms Flanking Your Sequence Being Inserted
		3.4 Synthesizing sgRNAs for Genome Editing
			3.4.1 Directly Ordering sgRNA or crRNA/tracrRNA from a Company
			3.4.2 Synthesizing sgRNAs from a DNA Template Using In Vitro Transcription
			3.4.3 Creating a New Plasmid Expressing Your sgRNA of Interest in the Worm
		3.5 Generating Homology-Directed Repair Templates
			3.5.1 Suggestions for Preparing Linear Double-Stranded Templates with 35 or 120 bp Homology Arms
			3.5.2 Generating Homology-Directed Repair Templates Using our Dual-Marker
				Selection Cassette Toolkit
		3.6 Preparing Injection Mixes for Microinjection
			3.6.1 Purified Ribonucleoprotein with Linear Double-Stranded DNA
				Homology-Directed Repair Template
			3.6.2 Purified Ribonucleoprotein with Plasmid Homology-Directed Repair Template
			3.6.3 All Plasmid Injection Mix
		3.7 Performing Microinjections
		3.8 Screening of Transgenic Progeny for Genome Edits
			3.8.1 Screening Directly for the Editing Event by Fluorescence Microscopy or by PCR Genotyping (for Homology-Directed Repair W...
			3.8.2 Antibiotic Selection and Screening of Animals with the Dual-Marker Cassette
		3.9 Excision of Dual-Marker Cassette by Cre Recombinase
	4 Notes
	References
Chapter 12: Targeted and Random Transposon-Assisted Single-Copy Transgene Insertion in C. elegans
	1 Introduction
	2 Materials
		2.1 MosSCI Insertion Strains
		2.2 MosSCI Targeting Vectors
		2.3 miniMos Targeting Vectors
		2.4 Co-injection Markers
	3 Methods
		3.1 MosSCI and miniMos Injections
		3.2 Isolation of Transgenic Animal with Single-Copy Insertion
		3.3 Validation of Single-Copy Transgene Insertion
		3.4 Identification of miniMos Insertion  Site
	4 Notes
	References
Chapter 13: Mutation Mapping and Identification by Whole-Genome Sequencing
	1 Introduction
	2 Materials
		2.1 Worm Growth and Mating
		2.2 gDNA Isolation
		2.3 Library Construction
		2.4 Data Analysis
	3 Methods
		3.1 Mating and F2 Selection (See Note 5)
		3.2 gDNA Isolation
		3.3 Library Construction
			3.3.1 End Prep Reaction
			3.3.2 Adapter Ligation Reaction
			3.3.3 Paramagnetic-Bead Clean-up (See Note 19)
			3.3.4 PCR Amplification
			3.3.5 Final Paramagnetic Bead Clean-up (See Note 23)
		3.4 Data Analysis
			3.4.1 Installation and Configuration (See Note 25)
			3.4.2 Pre-processing of Sequence  Data
			3.4.3 Alignment of Pre-processed Data to the C. elegans Reference Genome
			3.4.4 Post-processing of the Aligned Data for Variant Calling (Sort, Remove Duplicates, and Index)
			3.4.5 Hawaiian SNP Calling for Mapping (See Note 33)
			3.4.6 Variant Calling for Candidate Mutations (See Note 34)
			3.4.7 Annotation of Candidate Mutations (See Note 35)
	4 Notes
	References
Chapter 14: Lipid Extraction and Analysis
	1 Introduction
	2 Materials
		2.1 Lipid Extraction
		2.2 Thin-Layer Chromatography
		2.3 FAMEs and Gas Chromatography
		2.4 NGM Agar Plates for Growth of C. Elegans
		2.5 Alkaline Hypochlorite Method to Isolate C. elegans Embryos
	3 Methods
		3.1 Lipid Extraction from C. elegans
		3.2 Thin-Layer Chromatography of C. elegans Lipid Extracts
		3.3 Preparation of Nematode Samples for Fatty Acid Methyl Esters (FAMEs)
		3.4 Preparation of Fatty Acid Methyl Esters (FAMEs)
		3.5 GC-MS Separation of FAMEs
	4 Notes
	References
Chapter 15: Isolating Caenorhabditis elegans from the Natural Habitat
	1 Introduction
	2 Materials
		2.1 Collection and Storage of Substrate Samples
		2.2 Nematode Isolation in the Laboratory
		2.3 Morphological and Molecular Identification of C. elegans
		2.4 Genetic Identification Through Crosses with Established C. elegans Strains
		2.5 Establishment and Cryopreservation of C. elegans Wild Isolate Stocks
	3 Methods
		3.1 Finding Suitable Habitats and Substrate Samples
		3.2 Collection and Storage of Substrate Samples
		3.3 Nematode Isolation in the Laboratory
		3.4 Morphological and Molecular Identification of C. elegans
		3.5 Genetic Identification Through Crosses with Established C. elegans Strains
		3.6 Establishment and Cryopreservation of C. elegans Wild Isolate Stocks
	4 Notes
	References
Chapter 16: Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Microfluidic Device Casting and Punching
		2.3 Microfluidic Device Accessories
		2.4 Solutions
		2.5 Experimental Setup
		2.6 Alternative Materials
		2.7 Materials for Rapid Prototyping of Adhesive Tape Molds
	3 Methods
		3.1 Device Fabrication: PDMS Casting from a Mold Master
		3.2 Device Fabrication: Glass Substrates
			3.2.1 Glass Plates for Reusable Devices
			3.2.2 Plasma Bonding
		3.3 Device Preparation and Assembly
		3.4 Device Filling
		3.5 Worm Loading and Device Operation
			3.5.1 Troubleshooting Device Operation, Common Problems, and Recommended Solutions
		3.6 Device Cleanup and Storage
		3.7 Microfluidic Devices for Microscopy and Neural Imaging
			3.7.1 Parallel Trap Array Device
			3.7.2 Neuronal Imaging Device
		3.8 Rapid Prototyping of Microfluidic Mold Masters by Xurography
	4 Notes
	References
Chapter 17: Neuronal Microsurgery with an Yb-Doped Fiber Femtosecond Laser
	1 Introduction
	2 Materials
		2.1 Microscope Assembly
		2.2 Laser Microsurgery
	3 Methods
		3.1 Laser Safety
		3.2 Microscope and Laser Setup (Fig. 1A)
		3.3 Laser Alignment
			3.3.1 Alignment Process
			3.3.2 Fine Alignment Test: ``Black Ink Slide´´
		3.4 Laser Microsurgery
		3.5 Data Analysis
	4 Notes
	References
Chapter 18: An Imaging System for Monitoring C. elegans Behavior and Aging
	1 Introduction
	2 Materials
	3 Methods
		3.1 Designing the Imaging System
		3.2 Constructing the Mechanical Components
		3.3 Assembling the Camera, Lens, and Light Source
		3.4 Alignment and Optimization
		3.5 Acquiring Image  Data
	4 Notes
	References
Chapter 19: Methods for Modulating and Measuring Neuromuscular Exertion in C. elegans
	1 Introduction
		1.1 Single Worm: For the Study of Locomotion and Associated Behaviors
		1.2 Long Distance Assay: Screening by Burrowing Ability
		1.3 Surfacing Assay: Comparing Muscular Strength in Different Populations
		1.4 Restricted Burrowing Assay: To Study Kinematics and Cell Activity
		1.5 Long-Term Burrowing Assays: Assessing Changes Associated with Exertion
	2 Materials
		2.1 Single Worm Assay
		2.2 Long Distance Assay
		2.3 Surfacing Assay
		2.4 Restricted Burrowing Assay
		2.5 Long-Term Burrowing Assay
	3 Methods
		3.1 Single Worm Assay
		3.2 Long Distance Assay
		3.3 Surfacing Assay
		3.4 Restricted Burrowing: Making Wells
		3.5 Long-Term Burrowing
	4 Notes
	References
Chapter 20: Recording and Quantifying C. elegans Behavior
	1 Introduction
	2 Materials
		2.1 C. elegans Growth and Maintenance
		2.2 Single-Worm Tracking on the openAutoScope
		2.3 Multi-Worm Tracking
	3 Methods
		3.1 Single-Worm Tracking on the openAutoScope
			3.1.1 Preparation
			3.1.2 Collect the Lawn Boundary Information
			3.1.3 Collect Worm Data
			3.1.4 Analyzing the Data
		3.2 Multi-Worm Tracking
			3.2.1 Preparation
			3.2.2 Exploration Assay
			3.2.3 Multi-Worm Optogenetics
			3.2.4 Tracking and Analysis
	4 Notes
	References
Chapter 21: Primer on Mathematical Modeling in C. elegans
	1 Introduction
	2 How Mathematical Models Are Built
		2.1 A Conceptual Model
		2.2 Developing a Quantitative Model
		2.3 Formulating a Tractable Mathematical Model
		2.4 Testing and Validation
		2.5 Using the Model to Address Research Questions and Guide Experiments and Applications
	3 Example: Modeling the Heat-Shock Response
	4 Is My Question Suitable for Mathematical Modeling?
	References
Index