التعافي من اسماك الزرد الكبار قلوب للتطبيقات عالية الإنتاجية
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
استخدام في النظام النموذجي الزرد لدراسة التنمية والتجديد، والمرض يتوسع نحو استخدام قلوب الكبار لتفارق الخلية وتنقية RNA، DNA، والبروتينات. كل من هذه التطبيقات مطالبة الانتعاش السريع لأعداد كبيرة من قلوب الزرد لتجنب الجينات التنظيمية والتمثيل الغذائي، والتغيرات الأخرى التي تبدأ بعد الموت. كما يطلب قلوب الزرد الكبار لدراسة بنية القلب لمجموعة متنوعة من المسوخ وللدراسة تجديد القلب. ومع ذلك، فإن تشريح القلب الزرد التقليدي بطيء وصعب ويتطلب الأدوات المتخصصة، مما يجعل نطاق واسع تشريح قلوب الزرد الكبار مملة. الطرق التقليدية أيضا المرفأ من مخاطر الإضرار القلب أثناء تشريح. هنا، نحن تصف طريقة لتشريح قلوب الزرد الكبار التي هي سريعة، استنساخه، ويحافظ على بنية القلب. وعلاوة على ذلك، لا يتطلب هذا الأسلوب الأدوات المتخصصة، وغير مؤلم لالزرد،لا يمكن أن يؤديها على عينات جديدة أو ثابتة، ويمكن أن يؤديها على الزرد الشباب مثل شهر واحد من العمر. النهج وصف يوسع استخدام الزرد الكبار للأبحاث القلب والأوعية الدموية.
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
في حين أساليب لتشريح القلب الزرد الكبار وقد وصفت، كانت هذه الأساليب تستغرق وقتا طويلا، وتسبب عادة الأضرار التي لحقت القلب أثناء تشريح. لإجراء التجارب حيث قد تكون هناك حاجة لعدد كبير من قلوب الكبار، و / أو عندما تجنب تدهور أنسجة القلب من المهم للتطبيقات المصب، والوقت ?…
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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