Rückgewinnung von Adult Zebrafisch-Herzen für Hochdurchsatz-Anwendungen
Summary
Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.
Abstract
Verwendung des Zebrafisch-Modellsystem für die Untersuchung der Entwicklung, Regeneration und Krankheit wird in Richtung der Anwendung erwachsenen Herzen für Zelldissoziationslösung und Aufreinigung von RNA, DNA und Proteinen erweitert. Alle diese Anwendungen fordern die rasche Erholung der eine erhebliche Anzahl von Zebrafisch-Herz genregulatorischen, Stoffwechsel und andere Veränderungen, die nach dem Tod beginnen zu vermeiden. Erwachsenen Zebrafisch Herzen sind auch zur Untersuchung von Herzstruktur für eine Vielzahl von Mutanten und zur Untersuchung der Herzregeneration erforderlich. Jedoch ist die herkömmliche Zebrabärbling Herz Dissektion langsam und schwierig und erfordert Spezialwerkzeuge, so dass großflächige Dissektion erwachsenen Zebrafisch Herzen mühsam. Traditionelle Verfahren bergen auch das Risiko einer Beschädigung des Herzens während der Präparation. Hier beschreiben wir eine Methode zur Präparation der erwachsenen Zebrafisch Herzen, schnell, reproduzierbar ist, und bewahrt Herzen Architektur. Außerdem funktioniert diese Methode nicht verlangen Spezialwerkzeugen, ist schmerzfrei für den Zebrafisch,kann auf frischem oder feste Proben durchgeführt werden und kann an Zebrafischen im Alter von 1 Monat alt durchgeführt werden. Die beschriebene Vorgehensweise erweitert die Verwendung von erwachsenen Zebrafisch für Herz-Kreislauf-Forschung.
Introduction
Zebrafish are an excellent model for studying heart development and human disease1,2. Specific advantages include the translucent nature of zebrafish embryos, the availability of many genetic mutants and transgenic reporter lines, and the availability of genome editing technologies. In addition to their advantages for studying early heart development, zebrafish are an ideal system for studying vertebrate heart regeneration3.
More recently, adult zebrafish are playing an important part in bioinformatics approaches to studying cardiovascular development and disease, due to their relatively large clutch size and relatively quick and inexpensive breeding compared to other vertebrate models. Promising techniques include ribosome profiling, RNA-Seq, and cell dissociation and FACS sorting4-7. However, for these techniques the quality of the data can depend on obtaining a large number of samples in a rapid, efficient, and reproducible manner, before gene regulatory, metabolic, transcriptional, and other changes occur.
Dissection of adult zebrafish organs has been described in the past8,9. However, previous approaches to dissection of the heart were slow, ran the risk of damaging the heart during dissection, required special tools, and/or required fixation of the zebrafish prior to dissection; for these reasons, past approaches to zebrafish adult heart dissection were not optimized for high-throughput applications and/or applications requiring fresh tissue.
Here, we describe a method for adult zebrafish heart dissection that is simple, fast, efficient, and reproducible, while preserving cardiac morphology. This method does not include cutting into the pericardial space and therefore does not risk damaging the heart during dissection. Instead, this method relies on anatomical landmarks of the zebrafish, and therefore, it is highly reproducible. This dissection method is also versatile in that it can be used on fresh or fixed fish, and on zebrafish as young as one month old. Finally, this method results in minimal suffering to the zebrafish because after anesthesia and/or rapid cooling, the fish is additionally decapitated and pithed in the course of the dissection procedure.
Protocol
Representative Results
Discussion
Während Verfahren zum Präparieren des erwachsenen Zebrafisch Herzen beschrieben worden sind, sind diese Methoden zeitaufwendig und häufig verursacht beim Präparieren von Schädigungen des Herzens. Experimente, wo eine große Anzahl von erwachsenen Herzen erforderlich sein, führen und / oder wenn die Vermeidung von Abbau von Herzgewebe ist wichtig für die Downstream-Anwendungen ist die Zeit unter Verwendung traditioneller Techniken Dissektion erforderlich unerschwinglich. Ebenso ist reproduzierbar erhalten unbesch?…
Declarações
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Dr. Shaun Coughlin for hosting the filming of this procedure in his laboratory, and for general support. R.A. was supported by the NIH (F32HL110489) and the Sarnoff Cardiovascular Research Foundation. S.R. was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG) and the American Heart Association (AHA). D.Y.R.S was supported by the NIH (RO1HL54737), the Packard Foundation, and the Max Planck Society.
Materials
| small tank for transporting fish | Aquaneering | ZHCT100 | |
| fish net | Petsmart | 36-16731 | |
| 250mL glass beaker | Kimble | 14005-250 | |
| 9cm polystyrene petri dish | Nunc | 172958 | |
| razor blade | Personna American Safety Razor Company | 94-120-71 | |
| two Dumont #5SF forceps | Fine Science Tools | 11252-00 | |
| dissecting microscope | Olympus | SZX16 | |
| Tricaine | Sigma | A-5040 | |
| plastic transfer pipette | Thermo Scientific | 202-20S | |
| gooseneck light source | Dolan-Jenner Industries, Inc | Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System | |
| fluorescent light source | Lumen Dynamics | X-Cite 120Q | optional |
| micro-scissors | Biomedical Research Instruments, Inc | 11-1000 | optional |
| RBC Lysis Buffer | eBioscience | 00-4333-57 | optional |
Referências
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