Preface
Hox Genes: A Fertile Interplay of Concepts and Methods
Extracting Information from Genome and Gene Sequences
Manipulating Genomes
Regulatory Landscapes and Topological Organization of Regulatory Elements
Recognizing Expression Patterns
Addressing Gene Function in Vertebrate Model Organisms
Addressing Gene Function in Non-model Organisms
Identifying the Genomic Distribution and Target Genes of Hox Proteins
Probing Protein-DNA Interactions
Identifying Protein Partners
From the Bench to the Clinic
References
Contents
Contributors
Part I: Hox Genes: From Discovery to Functional Analysis
Chapter 1: Discovery and Classification of Homeobox Genes in Animal Genomes
1 Introduction
2 Materials
2.1 Genome
2.1.1 Track 1 (Without a Publicly Available Genome Sequence)
2.1.2 Track 2 (with a Publicly Available Genome Sequence)
2.2 Mining for Homeobox Sequences
2.3 Alignment and Phylogeny Reconstruction
3 Methods
3.1 Genome Sequencing and Assembly
3.1.1 Genome Sequencing
3.1.2 Checking Coverage
3.1.3 Read Preparation
3.1.4 Genome Assembly
3.2 Mining Genome Sequence for Homeobox Candidates
3.2.1 Search the Genome: BLAST
3.2.2 Search the Genome: HMMER
3.2.3 Extract Homeobox Sequences
3.3 Classifying Homeobox Sequences
3.3.1 BLAST Against GenBank and HomeoDB
3.3.2 Companion Domains
3.3.3 Alignment and Diagnostic Characters
3.3.4 Phylogenetic Tree Reconstruction
4 Notes
References
Chapter 2: How to Study Hox Gene Expression and Function in Mammalian Oocytes and Early Embryos
1 Introduction
2 Materials
2.1 RNA Extraction
2.2 DNase Treatment
2.3 Reverse Transcription
2.4 Quantitative Polymerase Chain Reaction (qPCR)
2.5 RNA Silencing
2.6 Oocytes and Embryos Collection, Fixation, and Permeabilization
2.7 Immuno-fluorescence
3 Methods
3.1 RNA Extraction
3.2 DNase Treatment
3.3 Reverse Transcription
3.4 Quantitative Polymerase Chain Reaction (qPCR)
3.5 Poly(dT): Hexamers Comparison
3.6 Injection of Zygotes
3.7 Injection of Oocytes
3.8 Hox Protein Detection by Immunofluorescence
3.8.1 Oocytes and Embryos Collection, Fixation, and Permeabilization
3.8.2 Immuno-fluorescence Staining of Oocytes and Embryos
4 Notes
References
Chapter 3: Genetic Lineage Tracing Analysis of Anterior Hox Expressing Cells
1 Introduction
2 Materials
2.1 Mouse Lines
2.2 Dissection of Embryos or Hearts
2.3 Para-formaldehyde (PFA)
2.4 X-Gal Staining
2.5 Freezing Embryos or Hearts for Cryosectioning
3 Methods
3.1 Crossing Mice
3.2 Collecting Embryos and Samples
3.3 X-Gal Staining
3.4 Sample Treatment
3.5 Sectioning and Image Capture
4 Notes
References
Chapter 4: A Genetic Strategy to Obtain P-Gal4 Elements in the Drosophila Hox Genes
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 5: Hox Complex Analysis Through BAC Recombineering
1 Introduction
2 Materials
2.1 Vector Construction Materials
2.2 Targeting PCR Materials
2.3 Recombineering Materials
2.4 Colony PCR Materials
2.5 Digest Screen Materials
2.6 CRE Recombinase Induction Materials
2.7 Recombineering Capture Materials
2.8 End Trimming, Homing Endonuclease Site/Insulator Insertion Materials
2.9 Seamless Insertions/Deletions (Two-Step Recombination with sacB-Kan Cassette) Materials
2.10 BAC Transgenesis Materials
2.11 Additional Materials/Equipment/Software
3 Methods
3.1 Vector Construction
3.2 Targeting PCR
3.3 Recombineering
3.4 Colony PCR
3.5 Digest Screen
3.6 CRE Recombinase Induction
3.7 Recombineering Capture
3.7.1 Oligo Construction
3.7.2 Cassette Cloning
3.7.3 Vector Linearization
3.8 Recombination-Mediated End Trimming, Homing Endonuclease Site/Insulator Insertion: Reduction of Transgenic Array Effects via Insulators and Homing Endonucleases
3.9 Seamless Insertions/Deletions (Two-Step Recombination with sacB-Kan Cassette)
3.10 BAC Transgenesis
4 Notes
References
Chapter 6: The Genetics of Murine Hox Loci: TAMERE, STRING, and PANTHERE to Engineer Chromosome Variants
1 Introduction
2 Materials
2.1 Choosing loxP Sites
2.2 Choosing Transgenic Cre-expressor Lines
3 Methods
3.1 Verifying loxP Sites
3.2 Genome Engineering Techniques
3.3 Screening Procedures
3.4 Verification of Novel Alleles
4 Notes
References
Chapter 7: Topological Organization of Drosophila Hox Genes Using DNA Fluorescent In Situ Hybridization
1 Introduction
2 Materials
2.1 Kits for Probe Direct Labeling
2.2 Components for Egg Laying
2.3 Components for the Fixation of Whole Mount Embryos
2.4 Components for FISH Hybridization
2.5 Components for DAPI and Mounting
2.6 Components Specific to Immunostaining (If Applicable)
2.7 Components Specific to FISH in Cells
2.8 Lab Equipments
2.8.1 For FISH in Tissues
2.8.2 For FISH in Cells
2.9 Software
3 Methods
3.1 Probe Labeling
3.2 FISH in Drosophila Embryos or Larval Discs
3.2.1 Preparation of Whole Embryos
3.2.2 Preparation of Larval Imaginal Discs
3.2.3 Hybridization to Fixed Embryos or Discs
3.2.4 FISH Combined with Immunostaining ( See Note 16)
3.3 FISH in Drosophila Cells
3.3.1 Cell Immobilization on Slides ( See Note 18)
3.3.2 FISH Procedure on Cells
3.3.3 FISH Combined with Immunostaining on Cells
3.4 Microscopy and Analysis
4 Notes
References
Chapter 8: Mining the Cis -Regulatory Elements of Hox Clusters
1 Introduction
2 Methods
2.1 Mining Highly Conserved Cis -Regulatory Elements from Hox Clusters
2.2 Mining Elusive Cis -Regulatory Elements
2.3 Mining CREs with Epigenetic Information
2.4 Functional Assays for In Silico-Identified Potential CREs
3 Concluding Remarks
References
Chapter 9: Functional Analysis of Hox Genes in Zebrafish
1 Introduction
2 Materials
2.1 General Laboratory Materials and Reagents
2.2 Fish Crosses, Embryo Injections, and Growth
2.3 mRNA Synthesis, MO Preparation
2.4 Tol2 Transgenesis
3 Methods
3.1 General Protocol for Microinjection in Zebrafish
3.2 mRNA Injections
3.3 Morpholino Antisense Oligonucleotide Injections
3.4 DNA Injections for Transgenesis
3.4.1 Preparation of the pTol2-Transgene Vector
3.4.2 Preparation of the Transposase mRNA
3.4.3 Injection into 1–2-Cell-Stage Embryos
3.4.4 Validation of Transgene Insertion
3.5 Genome Engineering Technologies
4 Notes
References
Chapter 10: Transgenesis in Non-model Organisms: The Case of Parhyale
1 Introduction
2 Materials
2.1 Microinjection of Early-Stage Parhyale Embryos
2.2 Testing the Activity of DNA Transposons with Excision and Transposition Assays
2.3 Transposon-Based Stable and Transient Transgenesis in Parhyale
2.4 Conditional Heat-Inducible Misexpression of Hox Genes in Transient and Stable Transgenic Parhyale
3 Methods
3.1 Microinjection of Early-Stage Parhyale Embryos
3.1.1 Collection of Parhyale Embryos
3.1.2 Preparation of Needles for Microinjection
3.1.3 Preparation of Agarose Steps
3.1.4 Microinjections
3.2 Testing the Activity of DNA Transposons with Excision and Transposition Assays
3.2.1 Preparation of Plasmid DNA
3.2.2 Preparation of Capped mRNA
3.2.3 Preparation of the Microinjection Mix
3.2.4 Microinjections and Nucleic Acid Extraction
3.2.5 Minos Excision Assay
3.2.6 Minos Transposition Assay
3.3 Transposon-Based Stable and Transient Transgenesis in Parhyale
3.4 Conditional Heat-Inducible Misexpression of Hox Genes in Transient and Stable Transgenic Parhyale
3.4.1 PCR-Based Isolation of Parhyale Heat-Inducible cis -Regulatory Sequences
3.4.2 Analysis of cis -Regulatory Sequences with Reporter Constructs in Transgenic Parhyale
3.4.3 Characterization of the Parhyale Heat-Inducible System
3.4.4 Cloning, Expression, and Functional Analysis of Parhyale Hox Genes
4 Notes
References
Chapter 11: Tissue Specific RNA Isolation in Drosophila Embryos: A Strategy to Analyze Context Dependent Transcriptome Landscapes Using FACS
1 Introduction
2 Materials
2.1 Egg Collection
2.2 Embryo Dissociation
2.3 FACS
2.4 RNA Isolation
3 Methods
3.1 Embryo Collection
3.2 Embryo Dissociation ( See Note 4)
3.3 FACS ( See Note 18)
3.4 RNA Isolation ( See Notes 22 and 23)
4 Notes
References
Chapter 12: Hox Transcriptomics in Drosophila Embryos
1 Introduction
2 Materials
2.1 Drosophila Embryo Collection
2.2 Isolation of RNA from Drosophila Embryos
2.3 Double-Stranded cDNA (ds-cDNA) and cRNA Synthesis and Cleanup, cRNA Fragmentation
2.4 Agarose Gel Analysis
2.5 Hybridization, Washing, Staining, and Scanning
3 Methods
3.1 Drosophila Embryo Collection
3.2 Isolation of RNA from Drosophila Embryos
3.3 ds-cDNA Synthesis
3.3.1 First-Strand cDNA Synthesis
3.3.2 Second-Strand cDNA Synthesis
3.3.3 ds-cDNA Cleanup
3.4 cRNA Synthesis
3.4.1 In Vitro Transcription
3.4.2 cRNA Cleanup
3.5 cRNA Fragmentation
3.6 Agarose Gel Analysis
3.7 Preparation of Hybridization Cocktail and Hybridization
3.8 Washing, Staining, and Scanning of the Arrays
4 Notes
References
Part II: Hox Proteins: Mode of Action and Biomedical Applications
Chapter 13: Measuring Hox-DNA Binding by Electrophoretic Mobility Shift Analysis
1 Introduction
2 Materials
2.1 DNA Annealing, DNA Labeling, and EMSA Equipment (Fig. 1)
2.2 DNA Annealing, DNA Labeling, and EMSA Chemicals and Reagents
2.3 Preparation of 0.5× TBE, the EMSA Running Buffer
2.4 Preparation of 0.1 M DTT
2.5 Preparation of TE Buffer (for the Nick Column)
2.6 Preparation of DNA-Binding Buffer ( See Note 3)
2.7 Preparation of Ammonium Persulfate (APS) Solution
2.8 Preparation of 1 % Agarose
3 Methods
3.1 Annealing DNA Oligonucleotides (Oligos) ( See Note 4)
3.2 Labeling DNA Oligos
3.3 Pouring the Gel
3.4 Diluting Protein and DNA Stocks
3.5 Mixing the Binding Reactions
3.6 Loading and Running the Gel
3.7 Drying the Gel and Phosphorimaging
3.8 Data Analysis for Activity Assays
3.9 Data Analysis for Affinity Assays
4 Notes
References
Chapter 14: Chromatin Immunoprecipitation and Chromatin Immunoprecipitation with Massively Parallel Sequencing on Mouse Embryonic Tissue
1 Introduction
2 Materials
2.1 Mouse Embryo Dissection and Cross-Linking
2.2 Chromatin Extraction
2.3 Immuno-precipitation
2.4 DNA Extraction
2.5 Equipment
3 Methods
3.1 Mouse Tissue Dissection and Cross-Linking
3.2 Chromatin Extraction and Sonication
3.3 Immuno-precipitation and DNA Extraction
4 Notes
References
Chapter 15: ChIP for Hox Proteins from Drosophila Imaginal Discs
1 Introduction
2 Materials
2.1 ChIP Buffers
2.2 Protein-A Sepharose Slurry
2.3 Consumable and Equipment
3 Methods
3.1 Experimental Controls
3.2 Tissue/Cell Isolation
3.3 Chromatin Cross-Link
3.4 Nuclear Lysis and Sonication
3.5 Analyzing the Size of Sheared Chromatin
3.6 Antibody Binding and Chromatin Pull Down
3.7 Reversal of Cross-Links and DNA Purification
3.8 Detection of Target Enrichment
4 Notes
References
Chapter 16: SELEX-seq: A Method for Characterizing the Complete Repertoire of Binding Site Preferences for Transcription Factor Complexes
1 Introduction
2 Materials
2.1 Preparation of SELEX Library and Control EMSA Probe
2.2 DNA-Binding Reaction and EMSA
2.3 Isolation and Elution of Bound DNA
2.4 DNA Amplification and Preparation of Sequencing Library
2.5 Computational Analysis of the Data
3 Methods
3.1 Preparation of SELEX Library and Control EMSA Probe
3.2 DNA-Binding Reaction and EMSA
3.3 Isolation and Elution of Bound DNA
3.4 DNA Amplification and Preparation of Sequencing Library
3.5 Modeling the Biases in the Initial Pool Using a Markov Model
3.6 Determining the Effective Length of the DNA-Binding Site
3.7 Calculating Relative Affinities Through Relative Enrichment of Motifs
3.8 Refinement of Affinity Estimates
4 Notes
References
Chapter 17: DamID as an Approach to Studying Long-Distance Chromatin Interactions
1 Introduction
2 Materials
3 Methods
3.1 Fly Growth Conditions
3.2 Genomic DNA Preparation from Whole Flies
3.3 Dpn II Digestion of Genomic DNA
3.4 DNA Preparation and Digestion from Dissected Fly Parts
3.5 Selection of Oligonucleotides for Q-PCR
3.6 Calibration of DNA for Q-PCR
3.7 Q-PCR Reactions
3.8 Preparation of Data
3.9 Statistical Analysis
4 Notes
References
Chapter 18: cgChIP: A Cell Type- and Gene-Specific Method for Chromatin Analysis
1 Introduction
2 Materials
2.1 CRMs of Interest
2.2 CRMs and Promoters Tagged with lacO -Binding Sites and Fusion to lacZ
2.3 Fly Lines for Cell-Type-Specific Expression of Epitope-Tagged LacI
2.4 Fly Culture Components
2.5 Chromatin Preparation Buffers and Solutions
2.6 Immuno-precipitation Components, Solutions, and Apparatus
2.7 Semiquantitative and Real-Time PCR Components, Solutions, and Apparatus
3 Methods
3.1 Fly Strain Development and Culture
3.2 Embryo Collections and Fixation
3.3 Chromatin Isolation for cgChIP
3.4 Titration and Quality Control of Chromatin Preparation
3.5 Cell-Type- and Gene-Specific Looping of Cis-Regulatory Modules
3.6 Cell-Type- and Gene-Specific Double Immunoprecipitation
3.7 Internal Controls for Evaluation of cgChIP Results
4 Notes
References
Chapter 19: Bimolecular Fluorescence Complementation (BiFC) in Live Drosophila Embryos
1 Introduction
2 Materials
2.1 Cloning Vectors with Venus Fragments
2.2 Protein Production
2.3 Electromobility Shift Assays (EMSAs)
2.4 Fly Lines
2.5 Embryo Collection
2.6 Immunostaining
3 Methods
3.1 Determining the Best Suitable Fusion Topologies for BiFC
3.2 Assessing the Expression Level of Fusion Constructs In Vivo
3.3 BiFC in Live Drosophila Embryos
4 Notes
References
Chapter 20: Hox Protein Interactions: Screening and Network Building
1 Introduction
2 Materials
2.1 Interaction Screening by Yeast Two-Hybrid (Y2H) Assay and High-Throughput Yeast Two-Hybrid Assay (HT-Y2H)
2.1.1 Yeast Strains
2.1.2 Expression Vectors
2.1.3 Media and Reagents
2.1.4 Primers
2.1.5 ß-Gal Filter Assay
2.1.6 Robotic Instruments
2.2 Interaction Validation by Affinity Co-purification
3 Methods
3.1 Interaction Screening by Yeast Two-Hybrid Assay (Y2H)
3.1.1 Plasmid Design and Construction
3.1.2 Two-Hybrid Yeast Auxotrophy Complementation
3.2 Interaction Screening by High-Throughput Yeast Two-Hybrid Assay (HT-Y2H)
3.2.1 Preparation of Baits and Prey Clones
3.2.2 Removal of Autoactivating DB-ORFs
3.2.3 HT-Y2H Screening
3.3 Interaction Validation by Affinity Co-purification
3.3.1 Plasmid Design and Construction
3.3.2 Cell Transfection
3.3.3 Affinity Co-purification
3.4 Network Building
3.4.1 Cytoscape Download and Installation
3.4.2 Plugin Installation
3.4.3 Complete Genome and Proteome Database Import ( See Note 18)
3.4.4 Import Data from Databases Using PSICQUIC Universal Web Service Client
3.4.5 ID Conversion and Unification Using CyThesaurus-ID-Mapping ( See Notes 27 and 28)
3.4.6 Network Merging
3.4.7 Network Processing
3.4.8 Network Layout
3.4.9 Network Analysis ( See Note 37)
4 Notes
References
Chapter 21: Rational Drug Repurposing Using sscMap Analysis in a HOX-TALE Model of Leukemia
1 Introduction
1.1 Hox Genes in Leukemia
1.2 Mining Open-
1.3 In Vitro and In Vivo Models of Leukemia
1.4 Connectivity Mapping and sscMap
1.5 Validation of sscMap
2 Materials
2.1 General Materials
2.2 RNA Extraction
2.3 Complementary DNA (cDNA) Generation
2.4 Methocult™ Assays
2.5 Ex Vivo/In Vivo Assays
3 Methods
3.1 Identifying Drugs
3.1.1 Identifying a Gene Signature
3.1.2 sscMap
3.2 Validation of sscMap by RQ-PCR
3.2.1 RNA Extraction
3.2.2 Complementary DNA (cDNA) Generation
3.2.3 RQ-PCR
3.3 Ex Vivo Treatment
3.4 Methocult™ Colony-Forming Unit Assay
3.4.1 Initial Plating
3.4.2 Replating Assays
3.4.3 Staining Methocult™ Colonies
3.5 Transplantation of Ex Vivo-Treated A9M-L2 Cells
3.6 In Vivo Treatment of Leukemic Mice
3.7 Disease Monitoring
3.7.1 Gross Pathology
3.7.2 Collection of Leukemic/Normal Hematopoietic Cells
4 Notes
References
Index