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.

참고 : 항상 IACUC 또는 윤리위원회의 승인이 제브라 피쉬를 사용하여 실험 절차를 시작하기 전에 제자리에 있는지 확인하십시오. 1. 시약 및 설치 준비 <ol><li> 다음과 같은 솔루션을 준비합니다. 표준 제브라 피쉬 매뉴얼 (10)에서 이러한 모든 솔루션에 대한 조리법을 찾을 수 있습니다. <ul><li> 계란 물 500㎖ </li><li> 계란 물에 0.03 % Tricaine 200 ㎖ </li><li> 1X 인산염 완충 식염수 100 ㎖ (PBS) </li><li> RBC 용해 버퍼 (선택 사항) </li><li> 얼음에 1.5 ml의 미세 원심 튜브에 선택의 대상 버퍼 (정착액, 트리 졸, PBS 등) 주 : 피부와 접촉하면 정착액 및 트리 졸은 독성. 이 물질을 취급 할 때는 장갑을 착용 할 것. </li></ul></li><li> 해부 설치를 준비합니다 : <ol><li> 해부 현미경을 통해 구즈넥 광원을 배치합니다. 해부 현미경 무대에서 9cm 페트리 접시의 뚜껑을 놓고에 1X PBS의 얇은 층을 부어그것은 (요리 자체가 쉽게 해부 너무 깊은 때문에 뚜껑이 사용됩니다). </li><li> 해부 근처의 수조에 페트리 접시 자체 (안 뚜껑)을 놓고 그 안에 1X PBS의 약 15 ml에 붓는다. 절개 한 후 심장을 씻어 사용합니다. 또한, RBC 용해 버퍼를 사용합니다. </li><li> 장소 면도날, 두 집게, microscissors (옵션)의 쌍, 및 현미경 옆에 전송 피펫. 급속 냉각에 의한 안락사 선택하면 얼음의 컨테이너를 가져옵니다. </li><li> Tricaine 의해 안락사 선택하면 250 ㎖의 유리 비이커에 달걀 물 0.03 % Tricaine 200㎖를 붓고. 제브라 피쉬의 사체의 처분을위한 작은 생물 학적 가방을 준비합니다. </li></ol></li></ol> 2. Zebrafish의 준비 <ol><li> 고정 물고기를 들어, 현미경 설정에, PBS에 세척 고정 물고기를 가져온다. <ol><li> &gt; 10 분 동안 얼음과 급속 냉각하여 전체 성인 제브라 피쉬를 안락사. </li><li> 복막을 통해 피부에 구멍을 만들기 위해 집게를 사용하여(멀리 마음에서) 고정액의 침투를 돕기 다음 4 ° C에서 RT 또는 O / N에서 2 시간 동안 수정 버퍼 (10)에 4 % 파라 포름 알데히드에 넣어합니다. </li></ol></li><li> 또한, 신선한 물고기, 장소 물고기는 작은 저장 탱크에 안락사하고 현미경 설정에 가져다. 개별 시설의 물고기 프로토콜과 해부 물고기 마음의 다운 스트림 용도에 따라 Tricaine 물고기를 마취 또는 잘린 전에 급속 냉각에 의해 안락사. <ol><li> 물고기가 움직이지 않을 때까지 얼음 물에 물고기 그물과 장소에 약 5 분을 한 물고기를 선택, 얼음을 사용합니다. </li><li> 또한, 아가미의 움직임이 멈출 때까지 용액의 비커에 물고기 그물과 장소 하나 물고기를 선택, Tricaine를 사용합니다. </li></ol></li><li> 빠르게 꼬리 지느러미에 의해 안락사 물고기를 선택하고 1X PBS로 가득 차 있었다 페트리 접시 덮개에 옆으로 누워. razo를 사용하는 동안, 한 손으로 가슴 지느러미를 들어 올려 집게를 사용하여R 블레이드는 가슴 지느러미 (그림 1A)의 부착 후부, 다른 손으로 물고기를 목을 벨 수 있습니다. 현미경을 통해보고하거나 직접 현미경 무대에서 물고기를 시각화하여이 작업 단계를 수행합니다. 참고 : 갓 안락사 물고기 여전히 잘린 후 피합니다. </li></ol><img alt="그림 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> 그림 1. Zebrafish의 성인 심장 해부는 제브라 피쉬의 해부학 적 구조를 사용한다. (A)는 물고기 목을 벨 집게와 가슴 지느러미를 들어 올려 그림과 같이 빨간색 점선을 따라 날카로운 깨끗한 면도날을 사용합니다. (B), 생선 머리를 안정 물고기 입 동안 포셉 한 가지를 배치하려면 다른 타인의 눈을 통해 거짓말을하고, 복부면이 위를하고 집게의 두 타인 페트리 접시의 바닥에 안정되도록하고 물고기 머리를 켜십시오. (C) 무료 그렇게해야 사용EPS는 아가미 (화살표). (D)이 리프팅의 첨부 파일을 잘라, 지느러미 대동맥 내강 혈액 (화살표)를 나타내는 핑크 스트라이프 흰색 구조로 볼 수있다. <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 3. 심장을 해부하다 <ol><li> 현미경으로 생선 머리를 시각화. 다른 타인이 눈 (그림 1B)를 통해, 머리 외부에있는 동안 하나의 집게로, 뇌를 통해 물고기의 입에 집게 중 하나 타인을 배치하여 물고기의 머리를 안정. 집게의 두 타인 페트리 접시의 바닥에 대한 안정세를 확인합니다. 물고기 머리를 이런 식으로 잡아서 복부 표면 위쪽으로 향해야합니다. </li><li> 다른 손으로, 몸 (그림 1C 화살표)에 아가미의 복부 첨부 파일을 잘라 두 번째 집게를 사용합니다. 집게와 약간이 리프팅, Dorsal 대동맥은 대동맥 루멘 (그림 1D 화살표)에 혈액의 빨간 줄무늬와 흰색 구조로 아래에 나타납니다. 대동맥 곤란 위쪽으로 잡아 당겨 집게 등의 대동맥을 잘라; 대안 등의 대동맥을 잘라 microscissors를 사용합니다. </li><li> 이제, 지느러미의 기지에서 몸 연골을 포함하여, 그 기지에서 가슴 지느러미를 파악하기 위해 두 번째 집게를 사용하고이 들어 올립니다. 다른 가슴 지느러미와 반복합니다. 때때로, 마음은 가슴 지느러미와 함께 나오는, 그래서 마음을 폐기하기 전에 연결되어 있지 않은 확인이 조각을 검사합니다. 참고 : (때때로는 가슴 지느러미와 벗기 있지만)이 시점에서, 마음이, 눈에 보이는 그대로 남은 생선의 머리에 연결해야합니다. 심장은 그 형상, 그 핑크 색상 및 색소 심낭에 의해 포위 됨으로써 확인할 수있다. 물고기의 안락사가 신속하게 수행되기 때문에, 갓 안락사 물고기 마음은 여전히​​ SLO를 구타 할 수있다wly. </li><li> 양손 집게를 보유하고 무료 있도록, 먼저 집게로 물고기의 머리 가자. 멀리 심낭의 마음을 애타게 두 집게를 사용합니다. 나머지 심낭 멀리 당겨 동안 지느러미 대동맥의 나머지에 의해 마음을 잡고. 참고 : 고정 또는 신선한 여부, 심장 주위의 결합 조직이 더 부서지기 쉬운 반면 당기 부드러운 강인한. 따라서, 모든 주변의 심낭 조직은 심장이 그대로 남아 멀리 해부 할 수 있습니다. </li><li> 생물 학적 가방에 남은 물고기 시체를 버리십시오. </li></ol> 4. 다운 스트림 응용 프로그램에 대한 마음을 준비 <ol><li> 집게를 사용하여, 지느러미 대동맥 / bulbus 동맥의 가장자리에 마음을 잡고 1X PBS의 신선한 접시에 마음을 놓습니다. 또한, 전송 피펫을 사용합니다. 앞뒤로 PBS의 마음을 움직 약 10 배 가능한 한 많은 피를 씻어. </li><li> 응용 프로그램의 모든 possib을 제거하는 것이 중요하다있는르 혈액 세포는 동일한 전후 모션을 사용하는 대신에 PBS 15 ml의 용해 완충액으로 RBC 9cm 페트리 접시에 씻는다. 심장의 구조를 유지하는 (예를 들어, RNA 제조), 심장 공동을 열고 멀리 혈구 린스 용이하게 집게 나 microscissors를 사용 하류 애플리케이션에 중요하지 않은 경우. </li><li> 별도의 심장 챔버가 요구되는 응용 프로그램의 경우, 서로 떨어져 bulbus 개존증, 심방 및 심실을 해부 집게 또는 microscissors를 사용합니다. </li><li> 얼음에 대상 버퍼에 심장, 또는 별도의 챔버를 전송합니다. 참고 : 일반 대상은 세포 분리, 4 % 파라 포름 알데히드, 또는 트리 졸에 대한 버퍼를 포함한다. </li></ol>

<ol>
	<li>Arnaout, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+model+for+human+long+QT+syndrome.">Zebrafish model for human long QT syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).</li><li>Chi, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system.">Genetic and physiologic dissection of the vertebrate cardiac conduction system.</a> PLoS Biology. 6 (5), 109 (2008).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Seminars in Cell &amp; Developmental Biology. 18 (1), 36-45 (2007).</li><li>Fang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Manoli, M., Driever, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+fluorescently+tagged+cells+from+zebrafish+larvae+for+RNA+isolation.">Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Cannon, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+the+haematopoietic+and+endothelial+transcriptome+during+zebrafish+development.">Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development.</a> Mechanisms of Development. 130 (2-3), 122-131 (2013).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. 37, (2010).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. 55, (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio. , (1993).</li></ol>

The authors have no disclosures.

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 ?...

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.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >small tank for transporting fish</td>
			<td >Aquaneering</td>
			<td >ZHCT100</td>
			<td></td>
		</tr>
		<tr >
			<td >fish net</td>
			<td >Petsmart</td>
			<td >36-16731</td>
			<td></td>
		</tr>
		<tr >
			<td >250mL glass beaker</td>
			<td >Kimble</td>
			<td >14005-250</td>
			<td></td>
		</tr>
		<tr >
			<td >9cm polystyrene petri dish</td>
			<td >Nunc</td>
			<td >172958</td>
			<td></td>
		</tr>
		<tr >
			<td >razor blade</td>
			<td >Personna American Safety Razor Company</td>
			<td >94-120-71</td>
			<td></td>
		</tr>
		<tr >
			<td >two Dumont #5SF forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-00</td>
			<td></td>
		</tr>
		<tr >
			<td >dissecting microscope</td>
			<td >Olympus</td>
			<td >SZX16</td>
			<td></td>
		</tr>
		<tr >
			<td >Tricaine</td>
			<td >Sigma</td>
			<td >A-5040</td>
			<td></td>
		</tr>
		<tr >
			<td >plastic transfer pipette</td>
			<td >Thermo Scientific</td>
			<td >202-20S</td>
			<td></td>
		</tr>
		<tr >
			<td >gooseneck light source</td>
			<td >Dolan-Jenner Industries, Inc</td>
			<td >Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System</td>
			<td></td>
		</tr>
		<tr >
			<td >fluorescent light source</td>
			<td >Lumen Dynamics</td>
			<td >X-Cite 120Q</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >micro-scissors</td>
			<td >Biomedical Research Instruments, Inc</td>
			<td >11-1000</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >RBC Lysis Buffer</td>
			<td >eBioscience</td>
			<td >00-4333-57</td>
			<td>optional</td>
		</tr>
	</tbody>
</table>

recovery of adult zebrafish hearts for high-throughput applications

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.

이 방법을 사용하여, 성인 심장 지브라 피쉬는 전통적인 방법을 사용하여 8 분에 걸쳐 5에 비해, 1 분 이하로 해부 될 수있다. 전통적인 방법 (8) 심낭에 맹목적으로 절단하기 때문에 일반적으로 심방 또는 bulbus 동맥 (그림 2B)의 손상이나 손실이 발생할 필요로하지만이 방법을 사용하여 해부 하트 (그림 2A) 안정적으로 그대로입니다. 해부 하트 그들?...

Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

Recovery of Adult Zebrafish Hearts for High-throughput Applications

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.

높은 처리량 응용 프로그램에 대한 성인 Zebrafish의 마음의 복구

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

recovery-of-adult-zebrafish-hearts-for-high-throughput-applications

Research

JoVE Journal

Biology

12.2K 조회수.  University of California San Francisco. 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.

비디오: Recovery of Adult Zebrafish Hearts for High-throughput Applications

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

높은 처리량 Zebrafish의 정자 Cryopreservation에 대한 방법과 체외에서 수정

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

연구

생물학

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

성인 Zebrafish에 Intraperitoneal 사출

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

직접 성인 Zebrafish의 신장에 세포의 이식

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

리그노셀룰로오스성 자료에 대한 높은 처리량 당화 분석

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Morpholinos의 생체내의 Electroporation에서 성인 Zebrafish의 망막에

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

생성중 성인 Zebrafish 테일 핀으로 Morpholinos의 생체내 Electroporation에

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of molecular events and therapeutic strategies as well as for evaluating the physiological mechanisms of some pathologies1. 
There are relatively few publications related to adult zebrafish physiology of organs and systems2, which may lead researchers to infer that the basic techniques needed to allow the exploration of zebrafish systems are lacking3. Hematologic biochemical values of zebrafish were first reported in 2003 by Murtha and colleagues4 who employed a blood collection technique first described by Jagadeeswaran and colleagues in 1999. Briefly, blood was collected via a micropipette tip through a lateral incision, approximately 0.3 cm in length, in the region of the dorsal aorta5. Because of the minute dimensions involved, this is a high-precision technique requiring a highly skilled practitioner. The same technique was used by the same group in another publication in that same year6. In 2010, Eames and colleagues assessed whole blood glucose levels in zebrafish7. They gained access to the blood by performing decapitations with scissors and then inserting a heparinized microcapillary collection tube into the pectoral articulation. They mention difficulties with hemolysis that were solved with an appropriate storage temperature based on the work Kilpatrick et al.8. When attempting to use Jagadeeswaran's technique in our laboratory, we found that it was difficult to make the incision in precisely the right place as not to allow a significant amount of blood to be lost before collection could be started. 
Recently, Gupta et al.9 described how to dissect adult zebrafish organs, Kinkle et al.10 described how to perform intraperitoneal injections, and Pugach et al.11 described how to perform retro-orbital injections. However, more work is needed to more fully explore basic techniques for research in zebrafish.
The small size of zebrafish presents challenges for researchers using it as an experimental model. Furthermore, given this smallness of scale, it is important that simple techniques are developed to enable researchers to explore the advantages of the zebrafish model.

This paper presents a technique for collection of blood from the dorsal aorta of Zebrafish. It also provides instructions for obtaining serum for use in biochemical analyses, such as tests for determining cholesterol and triglyceride levels.

성인 Zebrafish의 생물 화학 분석을위한 혈액 수집

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

성인 Zebrafish의를 Gavaging

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...