Drosophila: Methods and Protocols

Description

This third edition expands on the previous editions by providing updated protocols and current methods that cover recent breakthroughs in Drosophila research. Chapters guide readers through FlyBase, versatile gene expression systems, analysis of microRNA function, single-cell transcriptome data and metabolism, recent applications of CRISPR for precise genome editing, transcriptional activation and cell lineage tracing, protein inhibition and tagging, optogenetic and optochemical control of tissue mechanics, AFM measurements, sample preparation for electron microscopy, live imaging of different tissues and organs, and quantitative image analysis. Written in the format of the highly successful Methods in Molecular Biology series, chapters include an introduction to their topic, 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 cutting-edge, Drosophila: Methods and Protocols, Third Edition serves as a useful and practical guide to new researchers and experts using Drosophila as a model system. Preface Contents Contributors Chapter 1: Using FlyBase: A Database of Drosophila Genes and Genetics 1 Introduction 2 Overview 3 QuickSearch 3.1 Filtering Mixed Lists 3.2 GAL4 etc QuickSearch Tab 4 Controlled Vocabularies 5 The Gene Report 5.1 Naming, Classification, and Summaries 5.2 Functionality, Orthology, and References 6 Alleles and Phenotypes 6.1 Classical Alleles, Transgenic Constructs, and Insertions 6.2 Experimental Tools 6.3 Phenotypes 6.4 Disease Ontology Annotations 6.5 Disease-Implicated Variants 7 Expression Data 7.1 Low-Throughput Expression Data 7.2 High-Throughput Expression Data 8 Interactions 8.1 Physical Interactions 8.2 Genetic Interactions 9 Genomics Data 9.1 Gene Model Annotations in FlyBase 9.2 JBrowse 9.3 RNA-seq 10 Reagents 10.1 Stocks and Strains 10.2 Cell Lines 10.3 cDNAs 10.4 Antibodies 11 Integrated Reports 11.1 Large Dataset Metadata 11.2 Gene Groups 11.3 Pathways 11.4 Human Disease Models 12 Bulk Data Analysis and Downloads 12.1 Uploading and Analyzing Data 12.2 Downloading Data 12.3 Programmatic Access 13 Help Resources 14 The FlyBase Community 15 Alliance 16 The Future of FlyBase 16.1 Single-Cell RNA-seq 16.2 Chemical Curation 17 Concluding Remarks References Chapter 2: The Q-system: A Versatile Repressible Binary Expression System 1 Introduction 1.1 Origins of a Repressible Binary System: The Saccharomyces cerevisiae GAL Gene Cluster 1.2 Neurospora crassa qa Gene Cluster 1.3 Components of the Q-system 1.4 Modular Domains of Binary Expression Systems Enable Chimeric Transcription Factors 1.5 QF2, an Improved QF Transcription Factor for Transgenesis 2 Chimeric Transactivators and Split-QF 3 Temporal Control of Expression by Quinic Acid 4 Efficient Conversion of GAL4 Transgenes to QF2 Transgenes Using Homology Assisted CRISPR Knock-in (HACK) 5 Simultaneous Use of Multiple Binary Expression Systems and Intersectional Expression Utilizing the Q-system 6 Applications Beyond Drosophila 6.1 Mosquitoes 6.2 C. elegans 6.3 Zebrafish 6.4 Other Organisms 6.4.1 Plants 6.4.2 Fungi 6.4.3 Mammalian Cells 6.4.4 Bacteria 7 Future Directions References Chapter 3: Resources and Methods for the Analysis of MicroRNA Function in Drosophila 1 Introduction 2 Deletion Mutants 2.1 Applications 3 miRNA Sponges 3.1 Applications 4 miRNA Overexpression/Misexpression Strains 4.1 Applications 5 Target Identification and Analysis 6 Hybridization and Transgenic Methods to Detect Cell-Type Expression 6.1 Transcriptional Reporters 6.2 Sensors 6.3 In Situ Hybridization 7 Cloning Methods to Detect Cell-Type Expression 8 Concluding Summary References Chapter 4: Analysis of Single-Cell Transcriptome Data in Drosophila 1 Introduction 2 Laboratory Workflow 3 Data Analysis 3.1 Quality Control 3.2 Normalization 3.3 Finding Variable Features 3.4 Scaling and Regressing Out Biological or Technical Effects 3.5 Batch Correction and Data Integration 3.6 Dimensional Reduction and Clustering 3.7 Cluster Annotation Using Marker Genes 3.8 Possible Downstream Analyses 3.8.1 Gene Ontology and Network Analysis 3.8.2 Pseudotime Analysis and RNA Velocity 3.8.3 Machine Learning Approaches 3.8.4 Analysis of Base-Substitution Mutations 3.9 Computational Methods Developed Using Drosophila Single-Cell Data 4 Notes References Chapter 5: Prime Editing for Precise Genome Engineering in Drosophila 1 Introduction 2 Materials 2.1 Cloning pegRNA Expression Plasmids 2.2 S2R+ Cell Transfection (Optional) 2.3 Transgenic Fly Work 2.4 Detection of Edits 3 Methods 3.1 Locate and Annotate the Edit of Interest 3.2 Select a Prime Editing System 3.3 pegRNA/Nicking sgRNA Design 3.4 Cloning pegRNA Expression Plasmids 3.4.1 Cloning Annealed Oligos into pCFD3-NS 3.4.2 Cloning dsDNA into pCFD3-NS 3.4.3 Cloning dsDNAs into pCFD5-NS 3.5 Validation of Editing in S2R+ Cells (Optional) 3.6 pegRNA Transgenic Flies and Crossing 3.6.1 Isolation of pegRNA Transgenic Fly Lines 3.6.2 Prime Editing by Transgenic Crossing 3.7 Detection of Edits 3.7.1 Primer Design 3.7.2 Genomic DNA Extraction from S2R+ Cells or Single Flies 3.7.3 Amplicon Sequencing of S2R+ Cells or Somatic Editing Flies 3.7.4 Sanger Sequencing of Inherited Edits in Fly Lines 4 Notes References Chapter 6: CRISPR-/Cas9-Mediated Precise and Efficient Genome Editing in Drosophila 1 Introduction 2 Materials 2.1 Synthesis of sgRNAs 2.2 In Vivo Assay for Cleavage and NHEJ 2.3 Construction of Donor Plasmid 2.4 Purification of the Donor Plasmid 2.5 Cas9-Mediated Homologous DNA Repair 2.6 Excision of the DsRed Marker 3 Methods 3.1 Design and Synthesis of sgRNAs 3.2 Assay for Cleavage Efficiency of Synthesized sgRNAs 3.3 Design of the Donor Plasmid 3.4 Construction of the Donor Plasmid 3.5 Purification of the Donor Plasmid 3.6 Cas9-Mediated Homologous DNA Repair 3.7 Excision of the DsRed Marker with PiggyBac Transposase 4 Notes References Chapter 7: Tissue-Specific CRISPR-Cas9 Screening in Drosophila 1 Introduction 2 Materials 2.1 Transgenic Drosophila Strains Expressing Cas9 2.2 Transgenic sgRNA Lines 2.3 Fly Strains to Mark Cells with Active CRISPR-Cas9 2.4 Plasmids 2.5 Antibody Staining 3 Methods 3.1 Generation and Characterization of Gal4 UAS-Cas9 Fly Lines 3.1.1 Testing for Cas9-Mediated Toxicity 3.1.2 Testing for On-Target Efficiency of Gal4 UAS-uCas9 Lines 3.1.3 Analysis of Spatial Mutagenesis Patterns 3.2 Selection of Transgenic sgRNA Lines 3.2.1 The Heidelberg CRISPR Fly Design Library (Boutros Lab) 3.2.2 The TRIP Knockout Lines (Perrimon Lab) 3.2.3 The Weizmann Knockout Project Lines (Schuldiner Lab) 3.2.4 The NIG sgRNA Lines (Kondo and Ueda Lab) 3.2.5 Other Publicly Available sgRNA Collections 3.3 Controls and Experimental Conditions 3.3.1 Negative Controls 3.3.2 Positive Controls 3.3.3 Experimental Conditions 3.3.4 Pilot Screen 3.4 Confounding Factors 3.4.1 Effects That Can Give Rise to False-Negative Results Low Mutagenesis Efficiency mRNA and Protein Stability Silent Mutations and Genetic Compensation Genetic Mosaics 3.4.2 Effects That Can Give Rise to False-Positive Results Off-Target Mutagenesis Large Deletions and Genomic Rearrangements Loss of Heterozygosity 3.5 Strategies to Confirm Causality of On-Target Mutations 3.5.1 Perform Mutagenesis with Independent sgRNA Lines 3.5.2 Perturb Gene with Alternative Methods 3.5.3 Create Sequence Verified Germline Alleles 3.5.4 Rescue the Phenotype 4 Notes References Chapter 8: CRISPR-Based Transcriptional Activation in Drosophila 1 Introduction 2 Materials 2.1 Fly Stocks 2.2 Plasmid Construction Reagents 2.3 Injection Reagents 3 Methods 3.1 Backbone Plasmid (the flySAM Vector without sgRNA) Amplification 3.2 Backbone Preparation (Linearization by Enzyme Digestion) 3.3 Oligo Design 3.4 Ligation and Transformation 3.5 Cloning Multiple sgRNA into flySAM System 3.6 FlySAM Plasmid Purification and Microinjection 3.7 Transgenic Selection (for Detailed Procedure, see Fig. 3) 4 Notes References Chapter 9: Tracing and Manipulating Drosophila Cell Lineages Based on CRISPR: CaSSA and CLADES 1 Introduction 2 CaSSA 2.1 Rationale 2.2 A Simple Reporter 2.3 Refining Expression Patterns with GAL4 2.4 Finding New Genetic Drivers via CaSSA-Based Gene Trapping 2.5 Intersections and Unions 2.6 Notes 3 Clades 3.1 Key Concepts 3.2 The CLADES Construct 3.3 Controlling Cascade Progression 3.4 Notes 4 Analyzing Drosophila Cell Lineages with CaSSA and CLADES References Chapter 10: Studying Protein Function Using Nanobodies and Other Protein Binders in Drosophila 1 Introduction 2 Key Features of Protein Binder-Based Approaches 2.1 Availability of a Protein Binder 2.2 Availability or Generation of Targetable Proteins 2.3 Driving the Expression of Functionalized Binders 2.3.1 GAL4 System 2.3.2 Twists of the GAL4 System (GAL80, Split and Company) 2.3.3 Specific Enhancers and mRNA Regulatory Elements 2.4 Different Functionalizations of Protein Binders 2.4.1 Protein Degradation 2.4.2 Protein Trapping/Relocalization 2.4.3 Protein Visualization 2.4.4 Targeted Post-translational Modification 2.4.5 Recording Protein-Protein Interactions 2.4.6 Recording Protein-DNA Interactions 2.4.7 Recording Cell-Cell Interactions 3 Future Directions References Chapter 11: Anchor Away: A System for Fast Inhibition of Proteins in Drosophila 1 Introduction 2 Materials 2.1 Fly Lines, Fly Husbandry, and Dissection 2.2 Fusing FRB::GFP with the Target Gene 2.3 Protein Anchoring in Live Drosophila 2.4 Protein Anchoring in Cultured Wing Imaginal Discs 3 Methods 3.1 Fusing FRB::GFP with the Target Gene 3.2 Protein Anchoring in Live Drosophila 3.3 Protein Anchoring in Cultured Wing Imaginal Discs 4 Notes References Chapter 12: Tagging Drosophila Proteins with Genetically Encoded Fluorophores 1 Introduction 2 Available Fly Collections Expressing Fluorescently Tagged Proteins 2.1 Endogenously Tagged Genes 2.2 Tagged Transgenes 2.3 Advantages and Limitations of the Existing Fluorescently Tagged Fly Collections 3 Available Clone Collections 4 Methods to Fluorescently Tag a Protein of Choice in Drosophila 4.1 Tagging In Vitro by Recombineering 4.2 Large-Scale In Vivo Tagging with Generic Cassettes Using MiMIC or CRIMIC 4.3 In Vivo Tagging with CRISPR Using Gene-Specific Cassettes 5 Outlook 6 Notes References Chapter 13: Optogenetic Methods to Control Tissue Mechanics in Drosophila 1 Introduction 2 Materials 2.1 Sample Preparation 2.2 Microscopy 2.3 Fly Stocks 3 Methods 3.1 Fly Crosses 3.2 Embryo Collection and Mounting 3.3 Live Imaging 3.4 Global Photoactivation of the 5-Phosphatase OCRL1 3.5 Local Photoactivation of the 5-Phosphatase OCRL1 3.6 Local Photoactivation of RhoGEF2 4 Notes References Chapter 14: Optochemical Control of Cell Contractility in Drosophila Embryos 1 Introduction 2 Materials 2.1 Sample Preparation 2.2 Drosophila Strains 2.3 Microinjection 2.4 Ca2+ Uncaging 2.5 Imaging 2.6 Software 3 Methods 3.1 Microinjection Needle Preparation 3.2 Preparation of Flies 3.3 Preparation of Embryos 3.4 Microinjection 3.5 Uncaging Induces a Rapid Ca2+ Burst 3.6 Optochemical Control of Cell Contractility in the Columnar and Squamous Epithelia 3.7 Optochemical Control of Tissue Deformation 4 Notes References Chapter 15: Stiffness Measurement of Drosophila Egg Chambers by Atomic Force Microscopy 1 Introduction 2 Materials 2.1 Dish Preparation 2.2 Egg Chamber Dissection 2.3 AFM, Light Microscopy Setup 2.4 AFM Measurements on Egg Chambers 3 Methods 3.1 Dish Preparation 3.2 Egg Chamber Dissection 3.3 AFM Preparation 3.4 AFM Measurements on Egg Chambers 3.5 Data Analysis of AFM Measurements 3.6 Interpretation of Measured Apparent Young´s Moduli 4 Notes References Chapter 16: Cultivation and Live Imaging of Drosophila Imaginal Discs 1 Introduction 2 Materials 2.1 Culture Medium 2.2 Imaginal Disc Dissection 2.3 Mounting for Live Imaging 3 Methods 3.1 Preparation of the Larvae for Dissection 3.2 Dissection 3.3 Disc Culture Without Imaging 3.4 Preparation of the Live Imaging Chamber 3.5 Mounting of the Discs in the Live Imaging Chamber 3.6 Live Imaging 4 Notes References Chapter 17: Sample Preparation and Imaging of the Pupal Drosophila Abdominal Epidermis 1 Introduction 2 Materials 2.1 Live Imaging of Histoblasts and LECs 2.2 Live Imaging in Combination with Drug Application 3 Methods 3.1 Live Imaging 3.1.1 Cultivation of Properly Staged Pupae 3.1.2 Dissecting Staged Pupae 3.1.3 Mounting and Live Imaging 3.2 Live Imaging in Combination with Mosaic Analysis 3.2.1 Interchromosomal Recombination (Twin Spot Analysis and MARCM) 3.2.2 Overexpression Analysis with FLP-Out Clones 3.3 Live Imaging in Combination with Drug Application 3.3.1 Needle Preparation 3.3.2 Drug Injection 4 Notes References Chapter 18: FlyClear: A Tissue-Clearing Technique for High-Resolution Microscopy of Drosophila 1 Introduction 2 Materials 2.1 Tools for Sample Handling 2.2 Tools for Solution Measurements and Preparation 2.3 Fixative 2.4 Permeabilization 2.5 Depigmentation and Clearing Solution 2.6 Refractive Index Matching Medium if Mounted on Slides 2.7 Refractive Index Matching Solution if Mounted in a Cuvette 3 Methods 3.1 FlyClear Protocol (Third Instar Larva) 3.2 FlyClear Protocol (Prepupa) 3.3 FlyClear Protocol (Pupa) 3.4 FlyClear Protocol (Adult) 3.5 Mounting on Slides 3.6 Mounting in a Cuvette 4 Notes References Chapter 19: Preparation of Drosophila Tissues and Organs for Transmission Electron Microscopy 1 Introduction 2 Materials 2.1 Chemicals 2.2 Equipment 2.3 Safety Issues 3 Methods 3.1 Chemical Fixation of Drosophila Specimens 3.1.1 Preparation and Chemical Fixation of Embryos 3.1.2 Dissection and Chemical Fixation of Third Instar Wandering Larvae and Isolated Organs and Tissues 3.1.3 Dissection and Chemical Fixation of Pupae 3.1.4 Dissection and Chemical Fixation of Adult Flies 3.2 High-Pressure Freezing and Freeze-Substitution of Drosophila Specimens 3.3 Embedding of Chemically Fixed Embryos, Larvae, Pupae, or Adult Tissues 3.4 Trimming 3.5 Sections 3.6 Post-Staining (Contrasting with Heavy Metal Ions) 3.7 From Post-Staining to Transmission Electron Microscopy Images 3.8 Grids 3.9 Knives 4 Notes References Chapter 20: Segmentation and Quantitative Analysis of Epithelial Tissues 1 Introduction 2 Software 2.1 Software Installation 2.2 Run Tissue Analyzer 2.3 Input Data 3 Applications 3.1 Image Acquisition Guidelines 3.2 Image Pre-Processing 3.3 Automated Segmentation 3.4 Correction of Segmentation Masks 3.5 Cell Tracking 3.6 Correction of Cell Tracks 3.7 Data Analysis and Quantifications 3.8 Data Presentation 3.9 Going Further with Tissue Analyzer 4 Notes References Chapter 21: Genetically Encoded Sensors to Study Metabolism in Drosophila 1 Introduction 2 Materials 2.1 Equipment for Microscope Setup 2.2 Fly Stocks and Dissection Equipment 2.3 Buffers (See Note 5) 3 Methods (See Note 6) 3.1 Sample Preparation 3.2 Pump Setup 3.3 Buffer Exchange 3.4 Image Acquisition (See Note 14) 3.5 Data Analysis (See Note 15) 4 Notes References Index