Kooperativer Bibliotheksverbund

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Umfang: 1 online resource (632 pages) : , illustrations (some color).

Ausgabe: 4th ed.

ISBN: 9780128034880 , 0128034882 , 9780128034743 , 0128034742

Serie: Methods in Cell Biology ; Volume 135

Anmerkung: Front Cover -- The Zebrafish: Genetics, Genomics, and Transcriptomics -- Series Editors -- The Zebrafish: Genetics, Genomics, and Transcriptomics -- Copyright -- Dedication -- Contents -- Contributors -- Preface -- 01 - Forward and Reverse Genetics -- 1 - Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system -- INTRODUCTION -- 1. METHODS -- 1.1 Assembly of U6-Based sgRNA Transgenic Constructs -- 1.2 Construction of Cas9 Expression Vectors -- 1.3 Screening and Evaluation of Stable sgRNA or Cas9 Transgenic Fish -- 2. DISCUSSION -- SUMMARY -- Acknowledgments -- REFERENCES -- 2 - Tol2-mediated transgenesis, gene trapping, enhancer trapping, and Gal4-UAS system -- INTRODUCTION -- 1. TRANSGENESIS BY USING THE TOL2 TRANSPOSABLE ELEMENT -- 1.1 Rationale -- 1.2 Methods -- 1.2.1 Synthesis of transposase mRNA in vitro -- 1.2.2 Preparation of a Tol2-donor plasmid -- 1.2.3 Microinjection -- 1.2.4 Excision assay -- 1.2.5 Identification of transgenic fish -- 1.3 Materials -- 1.4 Discussion -- 2. GENE TRAP, ENHANCER TRAP METHODS FOR THE GAL4FF-UAS METHODS -- 2.1 Rationale -- 2.2 Methods -- 2.2.1 Gene trap and enhancer trap screens -- 2.2.2 Analysis of Tol2 insertions by Southern blot hybridization -- 2.2.3 Identification of Tol2 integration sites by inverse PCR -- 2.2.4 Search for useful gene trap and enhancer trap fish using the zTrap database -- 2.3 Materials -- 2.4 Discussion -- 3. TARGETED GENE EXPRESSION WITH THE GAL4-UAS SYSTEM -- 3.1 Rationale -- 3.2 Methods -- 3.2.1 Inhibition of neuronal activities via the Gal4-UAS system -- 3.2.2 Visualization of neuronal activities via the Gal4-UAS system -- 3.2.3 Visualization of in vivo microtubule structures via the Gal4-UAS system -- 3.2.4 Inhibition of the Wnt-signaling pathway via the Gal4-UAS system -- 3.3 Materials -- 3.4 Discussion -- Acknowledgments -- REFERENCES. , 3 - Genetic screens for mutations affecting adult traits and parental-effect genes -- INTRODUCTION -- EXTENDING THE RANGE OF FORWARD GENETICS ANALYSIS IN ZEBRAFISH SCREENS -- APPLICATION OF GENETIC SCREENS BEYOND THE EMBRYONIC LETHAL RANGE -- Post-embryonic development: juvenile and adults stages -- Prior to the midblastula transition: maternal- and paternal-effect genes -- 1. STRATEGIES FOR ADULT TRAIT AND PARENTAL-EFFECT SCREENS -- 1.1 F3 Screen for Adult Traits and F4 for Parental-Effect Traits Based on Natural Crosses That Integrates a Mapping Strategy -- 1.1.1 An F3-extended family approach with integrated mapping -- 1.1.2 Identification of maternal-zygotic, male sterile, and paternal-effect mutations -- 1.1.3 Assessment of the F4 natural crosses screen for maternal-effect mutations -- 1.2 F2 Adult or F3 Parental-Effect Trait Screens Based on EP-Induced Gynogenesis -- 1.2.1 Choice of gynogenetic method -- 1.2.2 Mutagenesis dose -- 1.2.3 Assessment of EP-based screens for maternal-effect mutations -- 1.3 Screening -- 1.3.1 Screening for adult traits -- 1.3.2 Screening embryos for parental-effects -- 2. SELECTION OF LINES FOR GENETIC SCREENS -- 2.1 Selection for Lethal/Sterile-Free Background Lines -- 2.1.1 Continuous inbreeding -- 2.1.2 Whole genome homozygosity through gynogenesis -- 2.2 Specific Requirements for Lines in Gynogenesis-Based Screens -- 2.2.1 Selection of lines that produce a high yield of gynogenotes -- 2.2.2 Selection for favorable sex ratios under gynogenetic conditions -- 2.3 A Hybrid/Inbred Approach -- 3. RECOVERY AND MAINTENANCE OF ADULT AND PARENTAL-EFFECT MUTATIONS -- 3.1 General Methods for Recovering Mutations -- 3.1.1 Recovery through known heterozygous carriers -- 3.1.2 Recovery through siblings of homozygous mutant individuals -- 3.1.3 Recovery through rare survivors. , 3.2 Maintenance of Adult and Parental-Effect Mutations -- 3.2.1 Maintenance of recessive lethal adult or parental-effect mapped mutations -- 3.2.2 Maintenance of recessive lethal adult or parental-effect unmapped mutations -- 4. MAPPING ADULT AND PARENTAL-EFFECT MUTATIONS, AND CLONING THE MUTANT GENE -- 4.1 Mapping and Mutant Gene Identification Using Next Generation Sequencing Technology -- 4.2 Mapping Concomitant With F3 Adult/F4 Parental-Effect Genetic Screens -- 4.3 Mapping After Identification and Recovery of Mutations -- 4.4 Efficient Fine Mapping of Maternal-Effect Mutations -- 5. SOLUTIONS, MATERIALS, AND PROTOCOLS -- 5.1 Solutions -- 5.2 Other Materials -- 5.3 Protocols -- 5.3.1 Sperm collection (adapted from D. Ransom) -- 5.3.2 UV-inactivation of sperm -- 5.3.3 Stripping of eggs -- 5.3.4 In vitro fertilization -- 5.3.5 Heat shock -- 5.3.6 Early pressure -- 5.3.7 Testosterone treatment -- CONCLUSIONS -- Acknowledgments -- REFERENCES -- 4 - Targeted candidate gene screens using CRISPR/Cas9 technology -- INTRODUCTION -- 1. GENERAL CONSIDERATIONS BEFORE STARTING A CRISPR SCREEN -- 2. CONSIDERATIONS FOR THE CAS9 ENZYME -- 3. CAS9 SYNTHESIS PROTOCOL -- 4. CONSIDERATIONS FOR CHOOSING SGRNA TARGET SITES -- 5. DESIGNING AN SGRNA -- 6. SGRNA DESIGN AND SYNTHESIS -- 7. SGRNA SYNTHESIS PROTOCOL -- 8. IDENTIFYING INJECTION CONDITIONS TO USE FOR THE SCREEN -- 9. ASSAYING THE RATE OF MUTAGENESIS IN F0-INJECTED EMBRYOS -- 10. CONFIRMATION OF PHENOTYPES -- 11. SUMMARY -- REFERENCES -- 5 - TALEN- and CRISPR-enhanced DNA homologous recombination for gene editing in zebrafish -- INTRODUCTION -- 1. MATERIALS -- 1.1 Reagents -- 1.2 Zebrafish and Microinjection -- 2. METHODS -- 2.1 In Vitro Synthesis of Cas9 mRNA -- 2.2 Preparation of CRISPR gRNAs -- 2.3 Microinjection of Cas9 mRNA/gRNA Into Zebrafish Embryos. , 2.4 Evaluation of CRISPR Efficiency in Injected Founder Embryos -- 2.5 Preparation of the Homologous Donor Template -- 2.6 Microinjection of Cas9 mRNA/gRNA and Donor Template -- 2.7 Detection of HR Events and Screening of Positive Founder Fish -- SUMMARY -- Acknowledgments -- REFERENCES -- 6 - Precise genome editing by homologous recombination -- INTRODUCTION -- 1. OVERVIEW OF CONTEMPORARY APPROACHES TO GENOME EDITING -- 2. SUMMARY OF WORKFLOW -- 3. FACTORS TO CONSIDER FOR GENOME EDITING VIA HOMOLOGOUS RECOMBINATION -- 3.1 Choice of Nuclease Target Site -- 3.2 Induction of Targeted DSBs -- 3.3 Design of Donor Sequences -- 3.4 Configuration of Donor Molecules -- 4. METHOD OF APPROACH -- 4.1 Design and Preparation of Programmed Nucleases -- 4.1.1 Design of nucleases -- 4.1.2 Target sequence confirmation and selection of a breeding population -- 4.1.3 Synthesis of programmable nucleases -- 4.1.4 Determination of nuclease cleavage activity -- 4.2 Design and Preparation of Donor Molecules Using pKHR Vectors -- 4.2.1 Preparation of homologous arms and novel donor sequences -- 4.2.2 Generating a loxP site within a homology arm -- 4.2.3 Assembling donor sequences into pKHR vectors -- 4.2.4 Sequence verification of donor plasmids -- 4.3 Microinjection of Programmed Nuclease and I-SceI-Digested Donor DNA -- 4.4 Detection of Recombination Events -- 4.5 Isolation of Founder Fish That Transmit Precisely Edited Genomes -- 4.5.1 Identification of F0 founders that transmit donor DNA sequences to progeny -- 4.5.2 Identification of founder fish carrying precisely edited genomes -- CONCLUSIONS -- Acknowledgments -- REFERENCES -- 7 - The zebrafish genome editing toolkit -- INTRODUCTION -- 1. REAGENTS -- 1.1 TALENs -- 1.2 CRISPR-Cas9 -- 1.3 Other Reagents -- 2. DNA REPAIR MECHANISMS-THE RISE OF MUTATIONS -- 2.1 Nonhomologous End Joining -- 2.2 Homology-Directed Repair. , 2.3 Microhomology-Mediated End Joining -- 3. MICROINJECTION -- 3.1 Injection Setup -- 3.2 Preparation of Reagents -- 3.3 Injection -- 3.4 Quality Control -- 4. SCREENING FOR F0 MUTANTS -- 4.1 Knock-ins -- 4.2 Knock-outs -- 5. SELECTING FOR DESIRABLE F1 MUTANTS -- 5.1 Outcross F0 Fish -- 5.2 Screen for Germline Transmissibility -- 5.3 F2 Generation and Onward -- CONCLUSION -- REFERENCES -- 8 - Clonal analysis of gene loss of function and tissue-specific gene deletion in zebrafish via CRISPR/Cas9 technology -- INTRODUCTION -- TISSUE-SPECIFIC KNOCKOUT IN ANIMAL MODELS -- CLONAL ANALYSIS OF GENE LOSS OF FUNCTION -- STATE OF THE ART IN ZEBRAFISH TISSUE-SPECIFIC GENE INACTIVATION -- CLONAL ANALYSIS OF GENE INACTIVATION IN ZEBRAFISH -- 1. METHODS FOR TISSUE-SPECIFIC GENE INACTIVATION AND CLONAL ANALYSIS OF MUTANT CELLS -- 1.1 Rationale for the Design of a Vector System for CRISPR/Cas9-Induced Conditional Gene Disruption via Gal4/UAS -- 1.2 Experimental Workflow -- 1.2.1 Overview of the method -- 1.2.2 Technical procedure -- 1.3 Rationale for the Design of a Vector System for Clonal Analysis of Mutant Cells by Combining the Gal4/UAS With the Cre/loxP ... -- 1.4 Experimental Workflow -- 1.5 Strategies for the Detection of Gene Loss of Function -- 1.5.1 Detection of protein loss in Cas9-expressing cells -- 1.5.1.1 Protocol for IHC on whole mount embryos -- 1.5.1.2 Protocol for IHC on cryosections -- 1.5.2 Molecular assessment of the mutagenesis efficiency via fluorescence-activated cell sorting and genome locus sequencing -- 1.5.2.1 Protocol for FACS -- 2. DISCUSSION -- REFERENCES -- 9 - Tissue-specific gene targeting using CRISPR/Cas9 -- INTRODUCTION -- 1. RATIONALE -- 1.1 Need for Tissue-Specific Tuning of Gene Expression -- 1.2 Limitations of Existing Gene-Silencing Methods -- 2. METHODS -- 2.1 Identification of CRISPR Target Sites. , 2.2 Construction of Vectors.

Sprache: Englisch