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.

注：常に動物実験委員会または倫理委員会の承認は、ゼブラフィッシュを使用して、任意の実験手順を開始する前に、所定の位置にあることを確認してください。 1.試薬およびセットアップを準備<ol><li>以下のソリューションを準備します。標準のゼブラフィッシュマニュアル10におけるこれらのソリューションのすべてのレシピを検索します。 <ul><li>卵水500mlの</li><li>卵、水で0.03％トリカインの200ミリリットル</li><li> 1×リン酸緩衝生理食塩水（PBS）100ml中</li><li> RBC溶解バッファー（オプション） </li><li>氷上の1.5mlマイクロチューブ中の選択肢の宛先バッファ（固定液、トリゾール、PBS など ） 注：彼らは皮膚に接触した場合に固定液とトリゾールは有毒である。これらの物質を取り扱う際は手袋を着用してください。 </li></ul></li><li>解剖のセットアップを準備します。 <ol><li>解剖顕微鏡上グースネック光源を配置します。解剖顕微鏡のステージ上で9センチメートルシャーレの蓋を置き、中に1×PBSの薄い層を注ぐそれ（ディッシュ自体は簡単に解剖のために深すぎるので、蓋が使用されている）。 </li><li>解剖スコープの近くにカウンター上のペトリ皿自体（いない蓋）を配置し、その中に1×PBSの約15ミリリットルを注ぐ。解剖後に心臓を洗浄するために使用します。また、RBC溶解バッファーを使用しています。 </li><li>置きかみそりの刃、顕微鏡の横に鉗子、microscissors（オプション）、および転送ピペットの2組。急速冷却による安楽死を選択した場合の氷のコンテナを取得します。 </li><li>トリカインによって安楽死を選択した場合250mlのガラスビーカーに卵水中の0.03％トリカインを200ml注ぐ。ゼブラフィッシュ死骸の処分のための小さなバイオハザードバッグを準備します。 </li></ol></li></ol> 2.ゼブラフィッシュを準備<ol><li>固定魚の場合は、顕微鏡のセットアップに、PBSですすぎ、固定された魚を、持って来る。 <ol><li> &gt; 10分間氷で急冷することによって全体の大人のゼブラフィッシュを安楽死させる。 </li><li>腹膜上の皮膚に穴を作るために鉗子を使用して、（心臓から）固定液の浸透を補助するため、その後、4℃でRTまたはO / Nで2時間修正バッファ10に4％パラホルムアルデヒドでそれらを置くために。 </li></ol></li><li>また、新鮮な魚のために、小さな保持タンクに安楽死させ、顕微鏡のセットアップにもたらすことが魚を置く。個々の施設の魚のプロトコルと解剖魚の心のために下流の用途に応じて、トリカインで魚を麻酔または断頭前に急速に冷却することにより安楽死させる。 <ol><li>魚は、約5分の移動を停止するまで、氷を使用するには、氷水で魚網と場所で1魚を拾う。 </li><li>鰓の動きが停止するまでまたは、トリカインを使用するために、溶液のビーカー内の魚網と場所で1魚を拾う。 </li></ol></li><li>すぐにそのテールフィンにより安楽死させた魚をピックアップし、1×PBSで満たしたペトリ皿のカバーでその側にそれを置く。 RAZOを使用しながら、片手で胸びれを持ち上げるために鉗子を使用して、Rもう一方の手で魚を斬首するブレード、ちょうど胸びれ（ 図1A）の結合を後方。顕微鏡を通して見ることによって、または単に直接顕微鏡ステージ上の魚を視覚化することによってどちらか、この手順を実行します。 注：新鮮な安楽死魚はまだ断頭後に出血します。 </li></ol><img alt="図1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> 図1.ゼブラフィッシュの大人の心臓解剖は、ゼブラフィッシュ、解剖学的ランドマークを利用しています。 （A）は 、魚を斬首ピンセットで胸びれを持ち上げ、図のように赤の点線に沿ってシャープなきれいなカミソリの刃を使用します。（B）、魚の頭を安定魚の口のしばらく鉗子の1歯を配置するには他の歯は、目全体に位置し、腹面がアップしていると鉗子の両方の歯は、ペトリ皿の底に対して安定しているように、その後魚の頭を回す。（C）無料FORCを使用してくださいEPSは蓋（矢印）。（D）は 、このリフティングの添付ファイルをカットする、背側大動脈を管腔血液（矢印）を示すピンクのストライプと白の構造として表示されている。 <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 3.ハートを解剖<ol><li>顕微鏡下で魚​​の頭を可視化。他の歯は、目（ 図1B）全体で、頭部の外にある間に1鉗子で、脳を通して、魚の口の中で鉗子の1歯を配置することにより、魚の頭を安定。鉗子の両方の歯は、ペトリ皿の底に対して安定していることを確認してください。魚の頭をこのように保持し、その腹面を上向きにする必要があります。 </li><li>もう一方の手で、体（矢印、 図1C）に蓋の腹側添付ファイルをカットする第二鉗子を使用しています。鉗子で少しこのリフティング、Dorsalの大動脈、大動脈内腔（矢印、 図1D）の血液の赤のストライプと白の構造体としての下に表示されます。大動脈をつまんで上向きに引いて、ピンセットで背側大動脈をカット。代わりに、背側大動脈をカットするmicroscissorsを使用しています。 </li><li>さて、フィンのベースのボディ軟骨を含む、そのベースから胸びれを把握することが第二の鉗子を使用し、これをオフに持ち上げます。他の胸鰭を繰り返します。時折、心臓は胸びれで出てくるので、心がそれらを廃棄する前に接続されていないことを確認するこれらの作品を調べます。 注：この時点で、心臓は（時にはそれが胸びれで外れが）まだそのまま、残りの魚の頭に接続され、表示されるはずです。心臓は、その形状、そのピンク色、および色素性心膜に囲まれた人間によって同定することができる。魚の安楽死を迅速に実行されているので、新鮮に安楽死魚の心はまだSLOを破っできるWLY。 </li><li>両手が鉗子を保持し、自由であるように、最初の鉗子で魚の頭を手放す。離れて心膜から心をいじめるために両方の鉗子を使用してください。残りの心膜が引き離されている間背部大動脈の残りの部分によって心をつかみます。 注：固定または新鮮かどうか、心が周囲の結合組織はよりもろい状態で引っ張って穏やかに堅牢である。そのため、周囲の心膜組織は、心臓が無傷で残っていると離れて解剖することができます。 </li><li>バイオハザードバッグ内の残りの魚の死体を捨てる。 </li></ol> 4.ダウンストリームアプリケーションのハートを準備<ol><li>鉗子を使用して、背側大動脈/動脈球の縁によって心をつかみ、1×PBSの新鮮な皿に心を置く。また、転送ピペットを使用しています。約10倍はできるだけ多くの血を洗い流すために、PBS中で前後に心を移動します。 </li><li>それはすべてpossibを除去することが重要である用途のためにルの血液細胞は、同一の往復運動を使用して、代わりにPBSを15mlのRBC溶解バッファーと9センチメートルシャーレに洗う。心臓の構造を維持すること（ 例えば、RNAする）、心臓腔を開いて離れ、血液細胞のすすぎ容易にするために、鉗子またはmicroscissorsを使用する下流側のアプリケーションのために重要でない場合。 </li><li>心腔を分離する用途では、所望の動脈球を分析するために鉗子またはmicroscissorsを使用し、心房、および互いに離間心室される。 </li><li>氷の上の宛先バッファに、心臓、または別々のチャンバを転送します。 注：一般的な目的地は、細胞解離、4％パラホルムアルデヒド、またはトリゾールための緩衝液を含む。 </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.

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...

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

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。

この方法を用いて、成体ゼブラフィッシュの心臓は、伝統的な方法8を用いて5分間かけて比較して、1分未満で解剖することができる。ハーツは、この方法を使用して解剖し、従来の方法8は心膜に盲目的に切断が必要なので、一般的にアトリウムまたは球部動脈（ 図2B）の損傷または損失の原因となりながら、確実に（ 図2A）は無傷である。切開した?...

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.

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

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 ビュー.  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.

ビデオ: 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.

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

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 ...

生物学

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.

アダルトゼブラフィッシュへの腹腔内注射

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.

直接アダルトゼブラフィッシュの腎臓への細胞の移植

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.

リグノセルロース系材料のためのハイスループット糖化アッセイ

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.

アダルトゼブラフィッシュ網膜にモルフォの in vivoエレクトロポレーションに

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.

リジェネアダルトゼブラフィッシュテールフィンにMorpholinos の in vivoエレクトロポレーションで

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.

大人のゼブラフィッシュの生化学的解析のための採血

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.

Gavagingアダルトゼブラフィッシュ

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.

ゼブラフィッシュ胚を用いた高スループットへのアプローチマニュアル小分子スクリーン

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.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

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