Vectorology for Optogenetics and Chemogenetics

Preface to the Series
About This Book
Contents
Contributors
Part I: Vectors Used in Systems Neuroscience Research
Chapter 1: Production, Testing, and Verification of Lentivirus for Regional Targeting in the Old-World Monkey Brain
1 Introduction
2 Lentivirus Production
3 Lentivirus Titer Determination and Stability Testing
4 Materials for Lentivirus Production
4.1 Solutions
5 Methods for Lentivirus Production
5.1 Cell Preparation
5.2 Transfection and Virus Collection
6 Method for Lentivirus Titering
6.1 RT-PCR (for VSV-G or FuG-E Lentivirus)
6.2 Transduction (for VSV-G Lentivirus Only)
References
Chapter 2: HiRet/NeuRet Vectors: Lentiviral System for Highly Efficient Gene Transfer Through Retrograde Axonal Transport
1 Introduction
2 Materials
2.1 Production of HiRet/NeuRet Vectors
2.2 Intracranial Injection of HiRet/NeuRet Vectors
3 Methods
3.1 Production of HiRet/NeuRet Vectors
3.2 Intracranial Injection of HiRet/NeuRet Vectors
4 Notes
References
Chapter 3: Generation of High-Titer Defective HSV-1 Amplicon Vectors
1 Introduction
2 Basic Protocol 1
2.1 Preparation of Helper Virus Stocks
2.1.1 Materials
2.1.2 Make a Plaque Plate
2.1.3 Make a Seed Stock
2.1.4 Amplify the Seed Stock
3 Support Protocol 1
3.1 Titration of Helper Virus by Plaque Assay
3.1.1 Additional Materials (also See Subheading 2)
4 Basic Protocol 2
4.1 Packaging Amplicon into Virus Particles
4.1.1 Materials
4.1.2 Prepare 2-2 Cells
4.1.3 Transfect Cells
4.1.4 Superinfect and Harvest Transfected Cells (P0) by Osmotic Lysis
4.1.5 Amplify Virus Stock (P1)
4.1.6 Amplify Virus Stock (P2)
4.1.7 Amplify Virus Stock (P3)
4.1.8 Purify and Concentrate the Virus
5 Support Protocol 2
5.1 Titration of Amplicon Virus by AMPLICON Vector Assay
5.1.1 Materials
5.1.2 Infect and Fix Cells
5.1.3 Record and Analyze Data
5.2 Reagents and Solutions
5.2.1 Alkaline Phosphatase (AP) Buffer
5.2.2 AP Substrate Solution
5.2.3 Crystal Violet Stain
5.2.4 Dulbecco´s Modified Essential Medium, Supplemented
5.2.5 Dulbecco´s Phosphate-Buffered Saline (D-PBS)
5.2.6 Paraformaldehyde Solution, 4% (W/V)
5.2.7 Plaque Agarose
5.2.8 Poly-d-Lysine Solution, 20 μg/mL
5.2.9 Sucrose Solution, 60%, 30%, and 10% (W/V)
6 Commentary
6.1 Background Information
6.2 Critical Parameters and Troubleshooting
6.2.1 Choosing the Right Helper Virus and Host Cell Combination
6.2.2 Optimizing Transfection and Infection Efficiencies
6.2.3 Optimizing the Packaging Procedure
6.2.4 Maintaining Virus Viability
6.3 Anticipated Results
6.4 Time Considerations
6.5 Packaging Schedule
References
Chapter 4: Generation and Application of Engineered Rabies Viral Vectors for Neural Circuit Research
1 Introduction
1.1 History of Neuroanatomy
1.2 Application of G-Deleted Rabies Virus (RABVΔG) for Neural Circuit Tracing
1.3 Overview and Rationale of Protocols
2 Materials
3 Step-by-Step Protocols for RABVΔG Production
3.1 Recovery of RABVΔG from cDNA
3.2 Amplification of RABVΔG
3.3 Pseudotyping of RABVΔG with EnvA
3.4 Concentration of the Viral Supernatant
3.5 Titration of Virus
4 Conclusion
References
Chapter 5: Quality Control for Adeno-Associated Viral Vector Production
1 Introduction
2 Materials
2.1 Titration by Quantitative PCR
2.2 Confirmation of Packaged AAV Genome by Identity PCR
2.3 Serotype Determination via Melting Temperature (AAV-ID)
2.4 Vector Purity by SDS-PAGE and Silver Staining
2.5 Limulus Amebocyte Lysate Chromogenic Endotoxin Test
2.6 In vitro Sterility and Expression Assay
3 Methods
3.1 Titration by Quantitative PCR
3.2 Confirmation of Packaged AAV Genome by Identity PCR
3.3 Serotype Determination via Melting Temperature (AAV-ID)-Protocol Adapted from Pacouret et al.
3.4 Vector Purity by SDS-PAGE and Silver Staining
3.5 Limulus Amebocyte Lysate Chromogenic Endotoxin Test
3.6 In vitro Sterility and Expression Assay
4 Notes
References
Part II: Vector Modifications and Applications
Chapter 6: Vector Tropism
1 Introduction
2 Adenoviridae (e.g., Ad5, HD-Ad, and CAV-2)
2.1 Adenovirus
2.2 Canine Adenovirus Type 2 (CAV-2)
3 Herpesviridae (e.g., HSV-1 and PRV)
3.1 Herpes Simplex Virus Type 1
3.2 Pseudorabies Virus(PRV)
4 Parvoviridae (e.g., AAV)
4.1 AAV Serotype Receptors and Tropisms
4.2 Delivery Routes and AAV Tropisms
4.2.1 AAV Intravenous Administration (IV)
4.2.2 Intraparenchymal Injection
4.2.3 Intracerebroventricular (ICV) Administration
4.3 Capsid Modification
4.3.1 Hybrid Capsids
4.3.2 Peptide Insertion
4.3.3 Capsid Shuffling and Directed Evolution
4.4 Regulatory Elements to Refine Gene Targeting
5 Retroviridae (e.g., Lentiviruses and Gamma-retroviruses)
6 Rhabdoviridae (e.g., SADB19-Rabies dG, dGL, SiR, and CVS-N2c)
7 Togaviridae (e.g., Sindbis Virus)
8 Summary
References
Chapter 7: Viruses for Systemic Delivery
1 Introduction: Adeno-associated Viral Vectors for Gene Transfer to the Nervous System
1.1 Engineered AAVs for Neuroscience Applications
1.2 Methodological Considerations for AAV Production and Use in Research
1.3 Procedural Overview
2 Materials
2.1 Triple Transient Transfection of HEK293T Cells
2.2 AAV Harvest
2.3 AAV Purification
2.4 AAV Titration
2.5 Systemic AAV Administration
3 Methods
4 Notes
References
Chapter 8: Transcriptomic Definition of Neuron Types
1 Introduction
1.1 Single-Cell Transcriptomics Overview
2 Material, Methods, and Results
2.1 Imaging and Surgery
2.2 Nuclei Isolation
2.3 Single Nucleus RNA Sequencing Library Preparation
2.4 Data Processing
2.5 Identification of MSN Clusters
2.6 MSN Subtype Annotations
2.7 Determining the Relationships Between MSN Subtypes
2.8 Interneurons in Primate Striatum
3 Conclusions and Discussions
References
Chapter 9: Enhancers for Selective Targeting
1 Introduction
2 Materials
2.1 Animals
2.2 scATAC-Seq
2.3 rAAV Production
2.4 Systemic Delivery in Mice
2.5 Local Delivery in Mice
2.6 PFA Perfusion
2.7 Vibratome Sectioning
2.8 Freezing Microtome Sectioning
2.9 Immunohistochemistry
2.10 In Situ Hybridization
2.11 Microscopy
3 Methods
3.1 Selection of Candidate Regulatory Elements
3.1.1 Selection of Genes Enriched in the Target Population
3.1.2 Cross-Species Conservation
3.1.3 scATAC-Seq
3.1.4 Selection of Candidate Enhancers
3.2 AAV Design/Production
3.2.1 rAAV Genome Vector Design
3.2.2 Transgene
3.2.3 Capsid
3.2.4 rAAV Production and Storage
3.3 Enhancer Screening Pipeline
3.3.1 Systemic Delivery
3.3.2 Local Delivery
3.3.3 PFA Perfusion and Brain Dissection
3.3.4 Vibratome Sectioning
3.3.5 Freezing Microtome Sectioning
3.3.6 Immunohistochemistry
3.3.7 In Situ Hybridization
3.4 Evaluation of Enhancer Expression Profile
3.4.1 Strength of Expression
3.4.2 Sensitivity and Specificity
4 Conclusion
References
Chapter 10: Pathway-Selective Reversible Perturbations Using a Double-Infection Technique in the Macaque Brain
1 Introduction
1.1 Injection of Viral Vectors
1.1.1 Targeting
1.1.2 Injections
1.1.3 Assessing Successful Delivery
1.2 Stimulation of Axon Terminals: Use of Anterograde Vectors
1.3 Stimulation of Retrogradely Transduced Cell Bodies: Use of Retrograde Vectors
1.3.1 Efficient Retrograde Vectors
1.3.2 Double-Infection Strategies
1.4 Limitations
1.5 Prospects
Glossary
References
Chapter 11: Pathway-Specific Chemogenetic Manipulation by Applying Ligand to Axonally Expressed DREADDs
1 Introduction
1.1 Why Choose DREADDs Over an Optogenetic Approach?
1.2 Targeting Neural Pathways with DREADDs
1.3 Disadvantages of Axonal DREADD Agonist Application to Control Pathways
2 Materials
2.1 Preoperative Supplies
2.2 Viral Injection Surgery Supplies
2.3 Virus Loading Supplies
2.4 Cannula Implantation Supplies
2.5 Postoperative Supplies
2.6 Microinjection Supplies
3 Protocol
3.1 Experimental Protocol for Targeting Pathways with Axon-Targeted DREADD Manipulations
3.1.1 Cannulae Implantation
3.1.2 Microinjection Procedures
4 Conclusion
References
Part III: Vector Delivery and Verification of Expression
Chapter 12: Convection Enhanced Delivery of Viral Vectors
1 Introduction
2 Materials
2.1 Baseline Imaging
2.2 Cannula Array Implantation
2.2.1 Reflux-Resistant Cannula
2.2.2 MR-Compatible Cannula Array and Chamber
2.2.3 Cannula Array Implantation
2.3 Viral Vector Delivery
2.4 Verification of Expression with Immunochemistry
3 Methods
3.1 Baseline Imaging
3.2 Cannula Array Implantation
3.2.1 Reflux-Resistant Cannula Fabrication
3.2.2 MR-Compatible Cannula Array and Cranial Chamber Fabrication
3.2.3 Cannula Array Implantation
3.3 Viral Vector Delivery
3.3.1 Preinfusion Preparation
3.3.2 MR-Guided Infusion
3.4 Verification of Expression with Immunohistochemistry
4 Notes
4.1 CED for Smaller Brains
4.2 Cannula Designs
4.3 Gel Models
4.4 Viral Vector Selection
4.5 Infusion Without Live MRI Monitoring
4.6 Targeting Deep Brain Structures
4.7 Cannula Insertion and Removal Speed
4.8 Flow Rates and Safety
5 Conclusion
References
Chapter 13: Multichannel Microinjector Arrays for Efficient Viral Vector Delivery into Rhesus Monkey Brain
1 Introduction
2 Multichannel Microinjector Array Assembly
2.1 Ventral 2 x 2 Array Assembly
2.2 Linear 1 x 4 Array Assembly
2.3 Linear 3 x 3 Array Assembly
3 Multichannel Microinjector Array Use
3.1 Array Use Protocol
3.2 Array Use Additional Notes
3.3 Manganese Contrast Visualization
3.4 Manganese-Virus Co-infusion Protocol
References
Chapter 14: Methods to Verify Expression and Function of DREADDs Using PET
1 Introduction
2 Materials
2.1 Animal Preparation
2.2 PET Scanner
2.3 PET Tracer and Drug Preparation
3 Methods
3.1 DREADD Expression Imaging by PET with [11C]DCZ
3.1.1 PET Scan
3.1.2 Data Analysis
3.1.3 Consideration of Off-Target Binding
3.2 DREADD Functional Imaging by PET with [18F]FDG
3.2.1 PET Imaging
3.2.2 Data Analysis
4 Notes
References
Chapter 15: Reporter Selection and Postmortem Methods to Verify Transgene Expression
1 Introduction
2 Tissue Preparation
3 Storage of Tissue
4 Preparation of Tissue for Light Microscopy and Fluorescence Microscopy
5 Blocking Aldehydes and Endogenous Signals
5.1 Aldehydes
5.2 Biotin
5.3 Peroxidase
5.4 Lipofuscin
6 Permeabilization and Blocking
7 Selection and Incubation of the Primary Antibody
8 Selection and Incubation of the Secondary Antibody
9 Overview of Different Detection Methods
9.1 Indirect Method
9.2 Avidin-Biotin Complex (ABC) Method
9.3 Peroxidase Anti-Peroxidase (PAP) Method
9.4 Immunogold Method
10 Example Protocols
10.1 Immunofluorescence Protocol
10.2 Light Microscopy ABC Immunoperoxidase Protocol
10.3 Pre-Embedding Electron Microscopy ABC Immunoperoxidase Protocol
11 Application Example: Use of EM Methods to Identify DREADDs
References
Chapter 16: Considerations for the Use of Viral Vectors in Nonhuman Primates
1 Introduction
2 Mechanical Considerations in NHP Experiments
2.1 Brain Size and Surgical Approaches
2.2 Using Cranial Chambers to Deliver Viruses to the Brain
2.2.1 Using Standard Cranial Chambers
2.2.2 The Use of Cranial Windows
3 Viral Considerations in NHP
3.1 Viral Capsid
3.1.1 Adeno-Associated Virus (AAVs)
3.1.2 Chimeric AAVs
3.1.3 Use of AAVs in Relation to NHP Experimentation
3.1.4 Lentiviral Vectors
3.1.5 Herpes Simplex Viral (HSV) Vectors
3.1.6 Rabies Viral Vectors
3.1.7 Canine Adenovirus Type 2 (CAV-2)
3.2 Viral Promoters and Enhancers
3.3 Interactions Between Capsids, Promoters, and Reporters.
3.4 Viral Reporters: Actuators and Indicators
4 Verification of Gene Transfer Efficacy in NHP
5 Summary
References
Index