Retinal Degeneration: Methods and Protocols (Methods in Molecular Biology, 1834)

✍ Scribed by Bernhard H. F. Weber (editor), Thomas Langmann (editor)

• Publisher: Humana
• Year: 2018
• Tongue: English
• Leaves: 413
• Category: Library

✦ Synopsis

This volume provides key updates on several methods from the first edition as well as including new novel techniques to address the most recent technological developments and their applications in retinal research Chapters guide readers through gene identification approaches, detailed protocols to generate functional retinal pigment epithelium cells, mouse retina and other animal models, fundus imaging and angiography, and cell-based treatment approaches. 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 cutting-edge, Retinal Degeneration: Methods and Protocols, Second Edition aims to ensure successful scientific work in the further study of this vital field.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Molecular Genetics and Tools
Chapter 1: Identification and Analysis of Genes Associated with Inherited Retinal Diseases
1 Introduction
1.1 Spectrum of Inherited Retinal Diseases
1.2 The Changing Landscape of Retinal Disease Gene Identification
1.3 Importance of Molecular Diagnostics
2 Techniques
2.1 Sanger Sequencing
2.2 Next-Generation Sequencing
2.3 Targeted Next-Generation Sequencing
2.4 Whole Exome Sequencing
2.5 Whole Genome Sequencing: Short-Read Versus Long-Read Techniques (Pros and Cons)
2.6 Homozygosity Mapping
2.7 Copy Number Variation Mapping
2.8 Linkage Analysis
2.9 In Vitro RNA Splice Assays
2.10 In Vivo Splice Studies Using Stem Cell Technology
2.11 Variant Data Interpretation
2.11.1 American College of Medical Genetics and Genomics
2.11.2 GeneMatcher
2.11.3 European Retinal Disease Consortium
2.12 Sequence Variant Registries and Databases
2.12.1 ClinVar
2.12.2 Leiden Open (Source) Variation Database
2.12.3 Human Gene Mutation Database
2.12.4 iEYE
3 Future Outlook
References
Chapter 2: Conduct and Quality Control of Differential Gene Expression Analysis Using High-Throughput Transcriptome Sequencing (RNASeq)
1 Introduction
2 Material
3 Installation
4 Methods
4.1 Download the Raw Reads from the GEO Database with the SRAToolkit
4.2 Prepare Reads and Conduct Basic Quality Control Steps
4.3 Run HISAT2 to Align the Reads to the Human Genome and Transcriptome
4.4 Quantify Transcript Abundance and Discover Novel Transcripts and Isoforms with the StringTie Program
4.5 Analyze the RNASeq Data with the Ballgown Library Implemented in R and Visualize the Results
5 Notes
References
Chapter 3: Testing for Known Retinal Degeneration Mutants in Mouse Strains
1 Introduction
2 Materials
2.1 Animals
2.2 Equipment
2.3 Reagents
3 Methods
3.1 DNA Extraction Hotshot Procedure
3.2 DNA Extraction Modified “Laird’s” Procedure
3.3 Genotyping Procedures
3.3.1 Retinal Degeneration 1
3.3.2 Retinal Degeneration 8
3.3.3 Retinal Degeneration 10
3.3.4 Retinal Degeneration 12
4 Notes
References
Chapter 4: CRISPR/Cas9 Gene Editing In Vitro and in Retinal Cells In Vivo
1 Introduction
2 Materials
2.1 Genomic DNA Extraction
2.2 Retinal Cell Dissociation
2.3 Site-Specific PCR and Bioinformatics Analysis
2.4 Retina Dissection
2.5 Western Blotting
3 Methods
3.1 Extraction of Genomic DNA from HeLa Cells
3.2 Dissociation of Retinal Cells
3.3 Extraction of Genomic DNA from Retinal Cells
3.4 Site-Specific PCR
3.5 Cloning of PCR Amplicons in TOPO TA Cloning Kit
3.6 Bioinformatic Analysis of PCR Amplicon Sequences
3.6.1 For Mac Users
3.6.2 For Window’s Users
3.7 Retina Dissection
3.8 Western Blotting on Dissected Retina
4 Notes
References
Chapter 5: Monitoring Surface Reactions by Combined Western Blot-ELISA Analysis
1 Introduction
2 Materials
2.1 ELISA
2.2 Cofactor Assay
2.3 Immunoblotting
3 Methods
3.1 ELISA
3.2 Cofactor Assay in Fluid Phase
3.3 Immunoblotting
3.3.1 Preparation of the Acrylamide Gels
3.3.2 SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis)
3.3.3 Immunoblotting Using Semidry Transfer Method
4 Notes
References
Part II: Cell/Tissue Culture Models
Chapter 6: Generation of Functional Retinal Pigment Epithelium from Human Induced Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Cell Differentiation and Cultivation Media
2.2 Cell Cultivation
2.3 TER Measurements
3 Methods
3.1 Differentiation of iPSCs
3.2 Cultivation of iPSC-Derived RPE
3.3 Long-Term Cryostorage of iPSC-Derived RPE in Liquid Nitrogen (−196 °C)
3.4 Functional Monitoring of iPSC-Derived RPE Monolayers by Measuring the Transepithelial Resistance (TER)
4 Notes
References
Chapter 7: Advanced Analysis of Photoreceptor Outer Segment Phagocytosis by RPE Cells in Culture
1 Introduction
2 Materials
2.1 POS Isolation Stock Solutions
2.2 POS Isolation Working Solutions
2.3 Solutions for POS Phagocytosis Assays
2.4 Equipment
3 Methods
3.1 Isolation of POS from Porcine Eyes
3.2 Plating of RPE Cells for Phagocytosis Assays
3.3 Continuous POS Phagocytosis Assay
3.4 Discontinuous POS Phagocytosis Assay with Synchronized Binding and Engulfment Steps
3.5 Quantification of Phagocytosed POS by Fluorescence Scanning
3.6 Quantification of Phagocytosed POS by Opsin Immunoblotting
3.7 Quantification of Phagocytosed POS by Fluorescence Microscopy
4 Notes
References
Chapter 8: Porcine RPE/Choroidal Explant Cultures
1 Introduction
2 Materials
2.1 Equipment
2.2 Culture Agents
2.3 Porcine Eyes
2.4 Drugs and Agents for Viability Assay
3 Methods
3.1 Preparation of Explants (Fig. 1)
3.2 Tissue Culture
3.3 Calcein-AM Viability Staining
4 Notes
References
Chapter 9: The Mouse Retinal Organoid Trisection Recipe: Efficient Generation of 3D Retinal Tissue from Mouse Embryonic Stem Cells
1 Introduction
2 Material
2.1 Cell Culture
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Mouse Embryonic Stem Cell Culture
3.1.1 Cell Thawing
3.1.2 Cell Passaging (See Note 10)
3.1.3 Cryopreservation (See Note 13)
3.2 Retinal Differentiation (See Note 15)
3.2.1 Day 0: Plating Cells for Retinal Differentiation
3.2.2 Day 1: Matrigel Supplementation
3.2.3 Day 7: Transfer to Retinal Maturation Medium
3.2.4 Day 10: Organoid Trisection (See Note 18)
3.2.5 From Day 14: Retinal Maturation
3.3 Making Cone- or Rod-Enriched Organoids
3.3.1 Cone-Enriched Organoids
3.3.2 Rod-Enriched Organoids
3.4 Cell Birth Dating
3.5 AAV Infection
3.6 Harvesting Organoids for  Immunohisto‑ chemistry
3.7 Harvesting Organoids for Gene Expression Analysis
3.7.1 Conventional Single or Bulk Organoid Gene Expression Analysis
3.7.2 Larger-Scale Single Organoid Gene Expression Analysis
3.8 Histology and Electron Microscopy of Retina Organoids
3.8.1 Histology of Methacrylate Resin Sections (Technovit 7100, [19])
3.8.2 Epon Embedding and TEM
4 Notes
References
Chapter 10: Cell Death Analysis in Retinal Cultures
1 Introduction
2 Materials
2.1 TUNEL on Retinal Explant Sections
2.1.1 Retinal Explant Culture
2.1.2 Fixation and Cryosectioning
2.1.3 TUNEL Assay
2.2 TUNEL on the 661W Photoreceptor-Like Cell Line
3 Methods
3.1 TUNEL on Retinal Explant Sections
3.1.1 Retinal Explant Culture
3.1.2 Fixation and Cryosectioning
3.1.3 TUNEL Assay
3.2 TUNEL on the 661W Photoreceptor-Like Cell Line
4 Notes
References
Chapter 11: Fate Mapping In Vivo to Distinguish Bona Fide Microglia Versus Recruited Monocyte-Derived Macrophages in Retinal Disease
1 Introduction
2 Materials
2.1 Generation of CX3CR1YFP − CreER/wt: R26tdTomato/wt Mice for Lineage Tracing
2.2 Tamoxifen Pulse Administration
2.3 Intravenous Leukocyte Exclusion Labeling
2.4 Digestion of Neural Retina
2.5 Staining for Flow Cytometry
2.6 Post-acquisition Analysis
3 Methods
3.1 Generation of CX3CR1YFP − CreER/wt: R26tdTomato/wt Mice for Fate Mapping
3.2 Tamoxifen Pulse Administration
3.3 Intravenous Leukocyte Exclusion Labeling
3.4 Digestion of Neural Retina
3.5 Staining for Flow Cytometry
3.6 Post-acquisition Analysis
4 Notes
References
Part III: Animal Models
Chapter 12: Light Damage Models of Retinal Degeneration
1 Introduction
2 Materials
2.1 Short-Term Exposure to High Levels of White Light
2.2 Short-Term Exposure to High-Intensity Blue or Green Light
3 Methods
3.1 Short-Term Exposure to High Levels of White Light
3.2 Exposure to Blue or Green Light
4 Notes
References
Chapter 13: Induction and Readout of Oxygen-Induced Retinopathy
1 Introduction
2 Material
2.1 Induction of Vaso-Obliteration
2.2 Perfusion with FITC-Labeled Dextran
2.3 Preparation of Retinal Whole Mounts and Isolectin B4 Staining
2.4 Analysis of Retinal Vasculature
3 Methods
3.1 Induction of Oxygen-Induced Retinopathy
3.2 Perfusion with FITC-Labeled Dextran
3.3 Preparation of Retinal Whole Mounts: Dissection
3.4 Isolectin B4 Staining
3.5 Preparation of Retinal Whole Mounts: Flat Mounting
3.6 Analysis and Quantification of Vaso-Obliteration and Superficial Retinal Vascularization
3.7 Analysis and Quantification of Retinal Neovascularization
3.8 Analysis and Quantification of Intraretinal Vessels
4 Notes
References
Chapter 14: Generation and Analysis of Xenopus laevis Models of Retinal Degeneration Using CRISPR/Cas9
1 Introduction
2 Materials
2.1 Generation of Cas9-Edited X. laevis Embryos
2.2 Dot Blot Analysis of Retinal Degeneration
3 Methods
3.1 Preparation of Cas9 mRNA and eGFP mRNA for Injection
3.2 Preparation of sgRNA for Injection
3.3 Generation of Cas9-Edited X. laevis Embryos
3.4 Analysis of Cas9-Editing of X. laevis Embryos by PCR
3.5 Dot Blot Analysis of Retinal Degeneration
4 Notes
References
Chapter 15: Gene Knockdown in Zebrafish (Danio rerio) as a Tool to Model Photoreceptor Diseases
1 Introduction
2 Materials
2.1 Zebrafish Maintenance and Breeding
2.2 Microinjection of Zebrafish Embryos
2.3 Analysis of the Zebrafish Retina by Immunofluorescence Staining
2.3.1 Fixation of Zebrafish Larvae
2.3.2 Embedding of Fixed Larvae
2.3.3 Cryosectioning
2.3.4 Immunofluo‑ rescence Staining of Cryosections
2.4 Single Larvae Western Blotting
2.5 Analysis of Visuomotor Behavior
3 Methods
3.1 Zebrafish Maintenance and Breeding
3.2 Microinjection of Zebrafish Embryos
3.3 Analysis of the  Zebrafish Retina by Immunofluo‑ rescence Staining
3.3.1 Fixation of Zebrafish Larvae
3.3.2 Embedding of Fixed Larvae
3.3.3 Cryosectioning
3.3.4 Immunofluo‑ rescence Staining of Retinal Sections
3.4 Single Larvae Western Blotting
3.5 Analysis of Visuomotor Behavior
4 Notes
References
Chapter 16: Drosophila melanogaster: A Valuable Genetic Model Organism to Elucidate the Biology of Retinitis Pigmentosa
1 Introduction
1.1 Comparison Between Drosophila and Vertebrate Retinal Structure and Function
1.2 Genetic Networks Conserved in Drosophila and Vertebrate Eye Development
1.3 Retinal Degeneration in Drosophila and Humans
2 Methods
2.1 Resources for Identification of Drosophila Disease Genes and Available Genetic Reagents
2.2 Generating a Disease Model Using Genetics
2.2.1 Forward Genetics
2.2.2 Reverse Genetics
2.2.3 Generation of Genetic Mosaics Using the Flp/FRT System
2.2.4 Targeted Expression Using the Gal4/UAS System
2.3 Important Aspects of Fly Culturing When Using the Retina as a Model System
2.3.1 Temperature
2.3.2 Diet
2.3.3 Light Conditions
2.3.4 Genetic Background
2.3.5 Age
2.4 Using the Disease Model to Identify Underlying Disease Mechanisms
2.4.1 Genetic Epistasis
2.4.2 “Omics”-Based Approaches
2.4.3 Using Drosophila Disease Models for Pharmacological Treatment
3 Conclusion
References
Part IV: Retinal Imaging
Chapter 17: Retinal Fundus Imaging in Mouse Models of Retinal Diseases
1 Introduction
2 Materials
2.1 Anesthesia
2.2 Pupil Dilation
2.3 Imaging
2.4 SPECTRALIS® HRA + Oct
2.5 Fundus Color Imaging with Visucam® 200
2.6 OCT Angiography with RTVue XR Avanti with AngioVue Device (Optovue Inc., Fremont, California, USA)
3 Methods
3.1 Anesthesia
3.2 Pupil Dilation
3.3 Positioning of the Animal in Front of the Laser/Imaging Device
3.4 Laser Treatment of the Fundus for CNV Induction
3.5 Autofluorescence Images
3.6 Infrared Image
3.7 Red-Free Image
3.8 Angiography
3.9 Optical Coherence Tomography (OCT)
3.10 OCT Angiography
3.11 Fundus Color Images
3.12 Mouse Models of Retinal Diseases
3.13 Mouse Models of Retinal and Choroidal Neovascularization
3.14 Mouse Models of Retinal Degeneration
4 Notes
References
Chapter 18: Phenotyping of Mouse Models with OCT
1 Introduction
2 Materials
2.1 OCT Device
2.2 Equipment to Adapt the OCT for Animal Use
2.3 Drugs
3 Methods
3.1 Device Modifications
3.2 Preparing the Animal
3.3 Software and Device Settings
3.4 Image Acquisition Settings
3.5 Data Analysis
4 Notes
References
Chapter 19: Cell-Specific Markers for the Identification of Retinal Cells and Subcellular Organelles by Immunofluorescence Microscopy
1 Introduction
2 Materials
2.1 Labeling of Retinal Cryosections for Immunofluorescence Microscopy
2.1.1 Fixation of Eyecup
2.1.2 Freezing Eyecup
2.1.3 Sectioning of Retina
2.1.4 Labeling of Retinal Cryosections
2.2 Labeling of Retinal Whole- Mount for Immunofluorescence Microscopy
2.2.1 Dissection and Marking of Retinal Whole-Mount
2.2.2 Separation of Retina from Eyecup
2.2.3 Labeling of Retinal Whole-Mount
2.2.4 Making Support for Whole-Mounting Retina on Glass Slides
2.2.5 Whole-Mounting Retina on Glass Slides
3 Methods
3.1 Labeling of Retinal Cryosections for Immunofluorescence Microscopy
3.1.1 Dissection of Retina
3.1.2 Cryo-Protection and Freezing of Retina
3.1.3 Sectioning of Retina
3.1.4 Labeling of Retinal Sections
3.2 Labeling of Retinal Whole- Mount for Immunofluorescence Microscopy
3.2.1 Dissection and Marking of Retinal Whole-Mount [33]
3.2.2 Separation of Retina from Eyecup [33]
3.2.3 Labeling of Retinal Whole-Mount
3.2.4 Making Support for Whole-Mounting Retina on Glass Slide
3.2.5 Whole-Mounting Retina on Glass Slide
4 Notes
References
Chapter 20: Immuno-TEM/STEM in Retinal Research
1 Introduction
2 Materials
2.1 Preparing Immuno-EM Blocks
2.2 Preparing Sections
2.3 Antibody Reaction
2.4 Staining and Carbon Coating
2.5 Imaging of Immuno-EM Samples
3 Methods
3.1 Sample Preparation, Day 1
3.2 Sample Preparation, Day 2
3.3 Sectioning
3.4 Antibody Reactions
3.5 Staining and Carbon Coating
3.6 Imaging with EM
4 Notes
References
Chapter 21: Noninvasive Two-Photon Microscopy Imaging of Mouse Retina and Retinal Pigment Epithelium
1 Introduction
2 Materials
2.1 Animals
2.2 Equipment
2.3 Supplies
3 Methods
3.1 Mice
3.2 Microscope Configuration
3.3 Adaptive Optics
3.4 Objectives
3.5 Imaging Ex Vivo
3.6 Imaging In Vivo
4 Notes
References
Chapter 22: Analysis of the Drosophila Compound Eye with Light and Electron Microscopy
1 Introduction
1.1 The Structure of the Drosophila Compound Eye
1.2 Various Phenotypes Associated with Distinct Mutations
1.3 Various Approaches to Study Drosophila Photoreceptors
1.4 Drosophila Genetics in Vision Research
2 Materials
2.1 Cryofixation, Reagents, and Equipment
2.2 Fixation for Light and Electron Microscopy
2.3 Section Preparation and Staining of Samples for Light Microscopy
2.4 Section Preparation and Staining of Samples for Electron Microscopy
2.4.1 Coating of Grids
2.4.2 Section Preparation and Staining of Grids
3 Methods
3.1 Section Preparation of the Compound Eye
3.2 Methods to Score for Rhabdomere Phenotypes in the Intact Head
3.2.1 Deep Pseudopupil (DPP) Analysis to Score for Defective Rhabdomeres
3.2.2 Optical Neutralization of the Cornea
3.3 Light Microscopy Approach
3.3.1 Tissue Fixation and Embedding for Light and Electron Microscopy
3.3.2 Dehydration and Embedding
3.3.3 Sectioning and Staining of the Eye for Light Microscopy
3.4 Electron Microscopy Approach
3.4.1 Electron Microscopic Analysis of Specimens
3.5 Cryofixation and Immunolabeling of Eye Sections for Confocal Microscopy
3.5.1 Cryosectioning
3.5.2 Cryo Labeling
3.6 Whole-Mount Staining for Confocal Microscopy
4 Notes
References
Part V: Therapies
Chapter 23: Cell-Based Therapy for Retinal Disease: The New Frontier
1 Introduction
2 Some Clinical and Preclinical Examples of Rescue
3 Some Preclinical Examples of Replacement
4 Challenges to Clinical Translation of Cell-Based Therapy
4.1 Cell Survival
4.2 Cell Delivery
4.3 Physiological Cell Behavior
4.4 Host Immune Response
4.5 Cancer Risk
5 Conclusion
References
Chapter 24: In Vitro Evaluation of AAV Vectors for Retinal Gene Therapy
1 Introduction
2 Materials
3 Methods
3.1 661W Cell Culture
3.2 661W Cell Transduction and Harvesting
3.3 Readout and Analysis
4 Notes
References
Chapter 25: Nanoparticles Targeting Retinal and Choroidal Capillaries In Vivo
1 Introduction
2 Materials
2.1 Particle Modification
2.2 Cell Culture
2.3 Microscopy of Cells and Tissue Sections
2.4 Animal Experiments
2.5 Tissue Preparation
3 Methods
3.1 Qdot Functionalization with RGD-Peptide
3.2 Quantification of Cellular Binding by Flow Cytometry
3.3 Analysis of Cellular Binding by Confocal Fluorescence Microscopy
3.4 Intravenous Administration in Mice
3.5 Preparation of Tissue Sections and Microscopic Analysis
4 Notes
References
Chapter 26: Optimized Subretinal Injection Technique for Gene Therapy Approaches
1 Introduction
2 Materials
2.1 Anesthesia, Solutions, and Agents
2.2 Surgical Instruments and Supplies
3 Methods
3.1 Preparation of Virus Suspension
3.2 Anesthesia and Preparation of Mice
3.3 Injection Procedure
3.4 Postoperative Treatment
4 Notes
References
Index