Description
The African turquoise killifish Nothobranchius furzeri is a short-lived fish species that has been established as a novel vertebrate model to study aging and represents a promising system to examine various other processes. The killifish is relatively easy and inexpensive to maintain. In addition, it has a short life cycle and abundant progeny, facilitating its use. Investigation using this model organism is growing and has provided new insights into aging, age-related diseases, ecology, evolution, and embryonic diapause—a state of “suspended animation.” This laboratory manual provides a comprehensive collection of protocols covering experimental techniques currently used to study the African turquoise killifish. Husbandry protocols describe how to set up and maintain a killifish colony and how to generate and preserve genetically engineered killifish lines. This collection also includes both general and specific methods to characterize different organs during aging, with a particular focus on the brain. Several genomics methods and analyses for unbiased characterization are also described. Finally, this manual encompasses protocols in other interesting areas of research, including injury repair, development, and embryonic diapause. This collection should enable studies in the African turquoise killifish and other species of killifish such as the South American killifish, and includes protocols that will be useful both to groups considering the use of these systems for the first time and to those with experience in the field. We also hope that this protocol book will foster a large killifish community and will promote new studies in many different research areas.
Contents
- Preface
- Anne Brunet
- OVERVIEW
- The African Turquoise Killifish: A Scalable Vertebrate Model for Aging and Other Complex Phenotypes
- Felix Boos, Jingxun Chen, and Anne Brunet
- PROTOCOLS
- Husbandry, Reproduction, and Preservation Protocols
- 1. Husbandry of the African Turquoise Killifish Nothobranchius furzeri
- Ravi D. Nath, Claire N. Bedbrook, Rahul Nagvekar, and Anne Brunet
- 2. Breeding and Reproduction of the African Turquoise Killifish Nothobranchius furzeri
- Jingxun Chen, Rishad C. Khondker, and Anne Brunet
- 3. Sperm Cryopreservation of the African Turquoise Killifish Nothobranchius furzeri
- Luca Dolfi, Tsz Kin Suen, Danel Medelbekova, Roberto Ripa, Orsolya Symmons, and Adam Antebi
- 4. In Vitro Fertilization of the African Turquoise Killifish Nothobranchius furzeri
- Luca Dolfi, Tsz Kin Suen, Danel Medelbekova, Roberto Ripa, Orsolya Symmons, and Adam Antebi
- 5. Noninvasive Ejaculate Collection from the African Turquoise Killifish Nothobranchius furzeri
- Silvia Cattelan and Dario Riccardo Valenzano
- Genetic Manipulation and Analysis
- 6. CRISPR–Cas9 Genome Editing in Nothobranchius furzeri for Gene Knockout and Knock-In
- Itai Rozenberg, Eitan Moses, and Itamar Harel
- 7. Efficient Transgenesis in African Turquoise Killifish Using a Gibson Assembly–Based Tol2 Transposon System
- Yufei Lou, Weifeng Lin, and Wei Wang
- 8. Rapid Genotyping of Nothobranchius furzeri Embryos, Larvae, and Adult Fish via High-Resolution Melt Analysis (HRMA)
- Johannes Krug, Annekatrin Richter, and Christoph Englert
- 9. Molecular Sexing of Nothobranchius furzeri Embryos and Larvae
- Annekatrin Richter, Johannes Krug, and Christoph Englert
- Genomics
- 10. Chromatin Accessibility Profiling and Data Analysis Using ATAC-seq in Nothobranchius furzeri
- G. Adam Reeves, Param Priya Singh, and Anne Brunet
- 11. Differential Expression Analysis of Nothobranchius furzeri Transposable Elements from RNA-seq Data
- Bryan Teefy, Matthew Malone, and Bérénice A. Benayoun
- 12. Taxonomic Profiling of Fecal and Intestinal Microbiota in the African Turquoise Killifish Nothobranchius furzeri
- Jens Seidel, Miriam Popkes, and Dario Riccardo Valenzano
- 13. Sequencing the Immunoglobulin Heavy-Chain Locus (IgH) in Turquoise Killifish (Nothobranchius furzeri)
- Jens Seidel, William John Bradshaw, and Dario Riccardo Valenzano
- Aging and Life Span Analysis
- 14. Life Span Assessment in the African Turquoise Killifish Nothobranchius furzeri
- Claire N. Bedbrook, Ravi D. Nath, Rogelio Barajas, and Anne Brunet
- 15. Molecular and Biochemical Analyses of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish
- Steven Bergmans, Sophie Vanhunsel, Caroline Zandecki, Eve Seuntjens, Lies De Groef, and Lieve Moons
- 16. Morphological Analysis of Aging Hallmarks in the Central Nervous System of the Fast-Aging African Turquoise Killifish
- Steven Bergmans, Sophie Vanhunsel, Jolien Van houcke, Valerie Mariën, Lut Arckens, and Lieve Moons
- Tissue Analysis
- General
- 17. Good Practices for Histological Analysis of the Annual Killifish Nothobranchius furzeri (Nothobranchiidae)
- Jakub Žák, Iva Dyková, Radim Blažek, and Martin Reichard
- Blood
- 18. Nonlethal Blood Sampling from the Killifish Nothobranchius furzeri
- Luca Dolfi, Roberto Ripa, Danel Medelbekova, Eugen Ballhysa, Orsolya Symmons, and Adam Antebi
- Fat
- 19. Micro-CT Analysis of Fat in the Killifish Nothobranchius furzeri
- Roberto Ripa, Andrea Mesaros, Orsolya Symmons, Eugen Ballhysa, Luca Dolfi, and Adam Antebi
- Brain
- 20. Long-Term Brain Organotypic Cultures of the Turquoise Killifish Nothobranchius furzeri
- Sara Bagnoli, Letizia Brogi, Baldassare Fronte, Carlo Bibbiani, Eva Terzibasi Tozzini, and Alessandro Cellerino
- 21. EdU and Immunofluorescence Staining of Nothobranchius furzeri Organotypic Cultures
- Sara Bagnoli, Eva Terzibasi Tozzini, and Alessandro Cellerino
- 22. Immunofluorescence and Aggresome Staining of Nothobranchius furzeri Cryosections
- Sara Bagnoli, Eva Terzibasi Tozzini, and Alessandro Cellerino
- 23. Whole-Brain Clearing and Immunofluorescence in Nothobranchius furzeri
- Sara Bagnoli, Eva Terzibasi Tozzini, and Alessandro Cellerino
- 24. Preparation of High-Viability Single-Cell Suspensions from African Turquoise Killifish Brain Tissue
- Valerie Mariën, Lutgarde Arckens, and Jolien Van houcke
- Nervous System Injury Methods
- 25. A Robust and Reproducible Method to Study Neurorepair after Stab Injury in the African Turquoise Killifish Telencephalon
- Valerie Mariën, Lutgarde Arckens, and Jolien Van houcke
- 26. The Optic Nerve Crush Injury Paradigm in African Turquoise Killifish to Study Axonal Regeneration in an Aged Environment
- Sophie Vanhunsel, Steven Bergmans, and Lieve Moons
- 27. Visually Driven Behavior Assays to Study Functionality and Recovery after Damage of the Aged African Turquoise Killifish Visual System
- Steven Bergmans, Sophie Vanhunsel, and Lieve Moons
- Embryo Development and Diapause
- 28. Handling Diapause Embryos of the African Turquoise Killifish Nothobranchius furzeri
- Chi-Kuo Hu
- 29. Fluorescent In Situ Hybridization in Embryos of the African Turquoise Killifish Nothobranchius furzeri
- Philip B. Abitua
- Appendix: General Safety and Hazardous Material Information
- Index
|
|
|
