恢复成年斑马鱼的心脏的高通量应用
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
用于研究发育，再生，和疾病正在扩大向着使用成年心脏细胞解离和RNA，DNA和蛋白质的纯化使用斑马鱼模型系统。所有这些应用都需要显著数字斑马鱼的心的迅速恢复，以避免基因调控，代谢和死亡后开始发生转变。也需要研究心脏结构，适用于各种突变体和研究心脏再生的成年斑马鱼的心。然而，传统的斑马鱼心脏解剖是缓慢而艰难的，需要专门的工具，使得成年斑马鱼心中大规模清扫乏味。传统的方法也怀有解剖过程中损坏心脏的风险。在这里，我们描述的方法对成年斑马鱼解剖心中是快速，可重复，并保留心脏结构。此外，这种方法不需要专门的工具，是无痛的斑马鱼，可以在新鲜的或固定的标本进行的，并且可以对斑马鱼年仅一个月大进行。所描述的方法扩展了使用成年斑马鱼的心血管研究。
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
而对于解剖成年斑马鱼心脏的方法已被描述的，这些方法是耗时的，而且经常造成夹层期间对心脏的损伤。执行实验中，可能需要大量的成人心，和/或当避免心脏组织的降解是用于下游应用重要，用传统的剥离工艺所需要的时间是令人望而却步。同样，重复获得完好的，完整的心是心脏结构的研究及免疫组化和显微镜方法很重要。我们发现，即使在初学者的手中，心切开的100％回收得到结构完整…
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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