높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구
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
개발, 재생을 공부하고, 질병이 세포 분리 및 RNA, DNA, 단백질의 정제를위한 성인 마음의 사용으로 확대되고의 제브라 피쉬 모델 시스템의 사용. 이러한 응용 프로그램의 모든 규제 유전자, 대사, 그리고 죽음 이후 시작하는 다른 변경을 방지하기 위해 제브라 피쉬 마음의 상당한 숫자의 신속한 복구를 요구하고있다. 성인 zebrafish의 마음은 돌연변이의 다양한 심장 구조를 공부하고 심장 재생을 공부해야합니다. 그러나, 기존의 제브라 피쉬의 심장 해부는 느리고 어려운 성인 zebrafish의 마음의 대규모 해부 지루한 제작, 특수 도구가 필요합니다. 전통적인 방법은 해부 동안 심장 손상의 위험을 항구. 여기, 우리는 빠른 재현, 심장 구조를 유지 성인 zebrafish의 마음의 절개하는 방법을 설명합니다. 또한,이 방법은 특수 도구가 필요하지 않습니다, 제브라 피쉬에 대한 통증이수 신선 또는 고정 시험편상에서 수행 될, 1 개월 이전처럼 어린 지브라 피쉬 수행 가능하다. 기재된 접근 방법은 심혈관 연구 성인 지브라 피쉬의 사용을 확장한다.
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
성인 zebrafish의 심장을 해부하는 방법이 설명되어 있지만,이 방법은 시간이 많이 소요되었고 일반적으로 해부하는 동안 심장에 손상을 일으키는 원인이되었다. 성인 하트 다수 필요할 수있는 실험을 수행하기 위해 및 / 또는 심장 조직의 열화를 피하는 하류 응용에 중요 할 때, 전통적인 절개 기술을 사용하여 요구되는 시간은 금지된다. 마찬가지로, 재현성 손상되지 않은 그대로의 마음을 얻는 ?…
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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