© 2024 • 286 pages, illustrated (48 color and 1 B&W), index
Paperback • £0.00 • ISBN 978-1-621824-60-2

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.

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