Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva
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
L'uso del sistema modello per lo studio zebrafish sviluppo, rigenerazione e malattia è espansione verso l'uso di cuori adulti per dissociazione cellulare e purificazione del DNA, RNA e proteine. Tutte queste applicazioni richiedono la rapida ripresa di un numero significativo di cuori zebrafish per evitare gene regolatore, metaboliche, e altri cambiamenti che iniziano dopo la morte. Cuori zebrafish adulti sono necessari anche per lo studio la struttura del cuore per una varietà di mutanti e per studiare la rigenerazione del cuore. Tuttavia, il tradizionale dissezione cuore zebrafish è lento e difficile e richiede strumenti specializzati, facendo su larga scala dissezione dei cuori di zebrafish adulto noioso. I metodi tradizionali porto anche il rischio di danneggiare il cuore durante la dissezione. Qui, si descrive un metodo per la dissezione di cuori di zebrafish adulto che è veloce, riproducibile, e conserva l'architettura del cuore. Inoltre, questo metodo non richiede strumenti specializzati, è indolore per il zebrafish,possono essere eseguite su campioni freschi o fissi, e possono essere eseguite sul pesce zebra giovane come un mese. L'approccio descritto espande l'uso di zebrafish adulto per la ricerca cardiovascolare.
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
Mentre sono stati descritti i metodi per sezionare cuore zebrafish adulto, questi metodi sono stati in termini di tempo e di solito causato danni al cuore durante la dissezione. Per eseguire esperimenti in cui può essere necessario un gran numero di cuori adulti, e / o quando evitare il degrado del tessuto cardiaco è importante per le applicazioni a valle, il tempo necessario utilizzando tecniche di dissezione tradizionali è proibitivo. Allo stesso modo, ottenendo riproducibile intatti, cuori intatti è importante pe…
Divulgazioni
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 |
Riferimenti
- Arnaout, R., et al. Zebrafish model for human long QT syndrome. Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).
- Chi, N. C., et al. Genetic and physiologic dissection of the vertebrate cardiac conduction system. PLoS Biology. 6 (5), 109 (2008).
- Poss, K. D. Getting to the heart of regeneration in zebrafish. Seminars in Cell & Developmental Biology. 18 (1), 36-45 (2007).
- Fang, Y., et al. Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration. Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).
- Manoli, M., Driever, W. Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation. Cold Spring Harbor Protocols. 2012 (8), (2012).
- Cannon, J. E., et al. Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development. Mechanisms of Development. 130 (2-3), 122-131 (2013).
- Wang, Z., Gerstein, M., Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics. 10 (1), 57-63 (2009).
- Gupta, T., Mullins, M. C. Dissection of organs from the adult zebrafish. Journal of Visualized Experiments. 37, (2010).
- Singleman, C., Holtzman, N. G. Heart dissection in larval, juvenile and adult zebrafish, Danio rerio. Journal of Visualized Experiments. 55, (2011).
- Westerfield, M. . The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio. , (1993).
