ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復
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
発達、再生、および疾患を研究するためのゼブラフィッシュモデル系の使用はRNA、DNA、およびタンパク質の細胞分離および精製のための大人の心の使用に向けて拡大している。これらのアプリケーションはすべて、調節遺伝子、代謝、および死後に始まる他の変更を避けるために、ゼブラフィッシュの心のかなりの数の急速な回復を要求する。大人のゼブラフィッシュの心も、突然変異体のさまざまな心臓の構造を研究するため、心臓の再生を研究するために必要とされる。しかし、従来のゼブラフィッシュの心臓解剖が遅いとは困難であり、退屈な大人のゼブラフィッシュの心の大規模な解剖をする、特殊なツールが必要です。伝統的な方法はまた、切開中に心臓を損傷する危険性を保有する。ここでは、高速で再現性があり、心臓のアーキテクチャを保持成体ゼブラフィッシュの心臓の解剖するための方法を記載する。さらに、この方法は、特殊なツールを必要としない、ゼブラフィッシュのために無痛である、新鮮なまたは固定された試料で行うことができ、生後1ヶ月の幼いゼブラフィッシュで行うことができる。記載されたアプローチは、心血管研究のための大人のゼブラフィッシュの使用を拡大する。
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
成体ゼブラフィッシュの心臓を切開するための方法が記載されているが、これらの方法は時間がかかりました、一般に切開の間に心臓への損傷を引き起こした。成人の心臓の多数必要になることが実験を実行するために、および/または心臓組織の劣化を回避することが下流の適用のために重要である場合、従来の切開技術を使用してかなりの時間が必要である。同様に、再現可能に損傷を受…
Disclosures
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 |
References
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