The authors have nothing to disclose.

すべての手順は、「実験動物管理の原則」で説明したガイドラインに従って実施された（厚生掲載の国民の協会がない。85から23、1985年改訂）し、承認されたロザリンド·フランクリン大学機関動物実験委員会の動物プロトコルは、8月19日。 有毛細胞壊死の1。ゲンタマイシン誘導<ol><li> 0.004％（4.32ミリモル）の最終濃度で通常の生理食塩水に硫酸ゲンタマイシンを準備します。 </li><li> 0.004％（4.32 mM）のゲンタマイシン溶液の入った容器に成魚（D.フィッシュ 、生後4〜6ヶ月）を配置します。すべてのコンテナを使用することができますが、我々は広い7、高における6、長期での7ですファーマ水生システムからの魚のコンテナを使用しています。 24時間28℃に設定したインキュベーター中で魚と容器を置きます。 24時間の期間の間生存可能な状態で魚を維持するのに十分なレベルで、タンク内の流体の総体積を設定する。注意：ゲンタマイシン流体の通気はNECEではありませんssary十分な量、治療される魚の数が使用される場合。 </li></ol>有毛細胞の2。生体染色<ol><li> 15 mg / mlとワーキングストック溶液からの蛍光生体色素[4-4 - ジエチルアミノスチリル）-N-メチルピリジニウムヨージド（485 nm励起λ、メタノール中で603 nm放射λ）（生理食塩水）で0.08％の濃度を準備しますエタノール中。 </li><li>ゲンタマイシン治療が有効であったかどうかを判断するために、制御およびゲンタマイシン処理魚のサブセットは、生体色素を含む6ウェル培養プレートのウェルに魚を置くことで、すぐに染色される。達成すべき統計的有意性のために必要とされる十分な魚の数（および培養プレートを使用してください。感丘カウントの検者速度に基づいて、魚は75オーバー分間染色されないように、時間をかけて、時間差をおいて、プレートに魚を置く工程2.3に記載のように。 </li><li> 、蛍光顕微鏡でベンチの引き出しにステップ2.2からプレートを配置染色neuromastsの検査に使用される。室温で1時間染色にわたって生体色素の消光を防止するために、部屋の照明をオフにします。 </li><li>色素ウォッシュアウトし、麻酔水タンクの両方を準備します。染料は水は通常の魚の水である、洗い流し、麻酔薬水のため、通常の魚の水中での1:1,000の希釈が達成されるよう、十分な2 - フェノキシエタノールを追加します。 </li><li>過剰生体色素をすすぎ、生体色素染色した魚の観察3.1に進み、過剰な通常の魚の水の中の魚を置きます。 </li><li> 28℃で8月16日の間に時間インキュベーターに通常の魚の水で洗浄し、ゲンタマイシン処理魚を移す、neuromastsの再生を検討するために、 </li><li> 8月16日時間の間に様々な時間で、魚が、培養器から取り出し、洗浄して、ステップ2.1から2.4に示したように染色する。生体染色色素で染色魚の観察3.1に進みます。 </li></ol> 3。麻酔魚とNeuromastsの蛍光カウント<ol><li&gt;ブロット紙タオル上の各魚が余分な水分を除去した後、プラスチックシャーレの蓋を中心とした濾紙の湿らせた一枚の上に配置する。 </li><li>ミッドボディステッチの生体色素染色neuromastsのデジタル画像を得るために、蛍光実体顕微鏡のステージ上にふたをする。 </li><li>使用する蛍光実体顕微鏡上に配置されたデジタルカメラは後続の定量分析のために画像を取り込むように2Xの倍率を設定する。実体顕微鏡の倍率設定が使用顕微鏡のブランドに依存してもよいが、設定はミッドボディのステッチ内で簡単に表示し、個々のneuromastsのカウントを許可する必要があります。注意して​​ください。 </li><li> （ 図1を参照）の右胸鰭のすぐ近くの魚の一番下の腹側の4指定された縫い目の中に見えるneuromastsの数を数えることによって回生量を決定します。統計分析のために、このような分散分析Oのような適切なテストを使用するRスチューデントのt検定。実験は、時点当たり5魚の最小値を利用するべきであり、全ての実験は最低3回繰り返されるべきである。 </li><li>感丘再生時間曲線に基づいて、（ 図3参照）、再生曲線の線形位相内であることが8〜16時間後ゲンタマイシンウォッシュアウトの間neuromastsを数える。注：線形時相の使用は、対照群と実験群との間の適切な定量分析を可能にする。 </li></ol>感丘分析が統計的に有意でない場合は定量分析より高い解像度を得るための個々の有毛細胞の4。蛍光カウント<ol><li> neuromastsのレベルの定量分析が有意でない場合、個々の有毛細胞のレベルでの分析は、より高い解像度を得るために利用することができる。特定の時点ポストゲンタマイシンウォッシュアウト（時点を以前感丘の研究に基づいて）、生体色素STで魚を選択するプロトコール2に記載し、その後1-5分間、1:500希釈で、2 - フェノキシエタノールを使用して、魚を安楽死させるように魚アイン。 </li><li>次のように正方形の皮膚弁の準備が行われているように、急冷を防ぐために、落ち着いた光の中で、4切開を行います。それは肛門フィンと整列するまで、魚の上部肋骨に沿って切開すると、腹全体で切開を行い、正方形の皮膚弁を作成したので、最後に、これらの切開の両側に二つの垂直切開を行う。注：この肌の準備が感丘の実験で使用したミッドボディステッチを組み込む予定。 </li><li>スライドガラス上の皮膚片を配置し、アンカーを助け、その後のデジタルイメージングのための組織をフラット化するために切除された皮膚試料上円形ガラスカバースリップを配置。 </li><li>ステップ4.3から皮膚片を使用して、ミッドボディーステッチの各感丘内有毛細胞のデジタル画像を取得する。少なくとも60Xの倍率で画像を撮影した後、髪のCEを数える対照群と実験群との比較定量分析のための個々のneuromasts以内LLS（ 図4参照）。 </li></ol>

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This work was supported by a research grant from the Iacocca Family Foundation, National Institutes of Health Grant DK092721 (to R.V.I.), and Rosalind Franklin University start-up funds. No potential conflicts of interest relevant to this article were reported. G.C.P., S.M.M, and N.D. researched data. M.P.S. Jr. and RI oversaw the project, contributed to the discussion of the data, and oversaw the writing and editing of the manuscript.

胚および幼虫ゼブラフィッシュ8,24,25の横ライン（LL）再生の解析のために確立されている多くの文献に基づいて、我々の研究の目的は、ゼブラフィッシュの横のライン再生のための定量的アッセイを開発することだったことでし最高の成魚で研究された疾患モデルに適用される。私たちは、成魚に胚/仔魚のために開発された手順を適用するときに、ある臨界点が重要であることがわ?...

側線（LL）システムは、聴覚、バランス、走流性や、学校教育や捕食の回避1-5として仲介行動する責任があり、両方の魚や両生類に見られる機械刺激器官である。これは、支持細胞によって取り囲ま有毛細胞のクラスターで構成され、両方ともがneuromasts 6と呼ばれる構造に配置されている。これらneuromastsは、一般的に魚の頭の中で観察されたいくつかの横のステッチと身体と尾の長軸に沿って垂直線（ステッチと呼ばれる）に編成されています。成人では、neuromastsは、胚や仔魚6と比較して、ステッチ内の数値が有意に大きい。ゼブラフィッシュにおける生物医学の研究では、抗生物質治療、ノイズによる外傷、慢性感染症などの影響に焦点を当てている。より良い人間にその効果を理解するための試みで、有毛細胞に7,8。 ほとんどの脊椎動物とは異なり、TEこのようなゼブラフィッシュ（ ゼブラフィッシュ ）などleostsは、失われた有毛細胞を再生する能力を持っている。ゼブラフィッシュは、それらの迅速な開発時間と高い再生能力が特に有用である。今日まで、しかし;側線の開発および/ ​​または再生にゼブラフィッシュの研究では、主に簡単にカウントおよび分析6,9,10を可能にし、横のラインneuromasts数の減少に胚および幼虫期の魚を利用している。 しかし、神経学的および非神経疾患11-16のような多くのゼブラフィッシュモデルは成魚としない幼虫で研究されて、我々はゲンタマイシンを使用して大人のゼブラフィッシュの横のラインに回生アッセイ（以前にゼブラフィッシュ幼生で使用されるアミノ配糖体の開発に焦点を当て、さらに最近では成魚17で使用される）ように、アッセイは、現在の大 ​​人のゼブラフィッシュの疾患モデルに適用することができるよう利用可能であった。ヴァントランプeだけしばらく以前に公開された手順Tら17は、成魚での有毛細胞の切除のための条件を確立し、それらはコントロールとそのようなトランスジェニックゼブラフィッシュ系統または薬理学的に誘発される疾患状態を使用している場合などの実験グループ間の定量的な比較のために必要とされる感丘再生のための標準曲線を確立できませんでしたゼブラフィッシュ18。したがって、我々は、対照群と実験群を比較した場合、そのような大人のゼブラフィッシュ疾患モデルと同じように我々のデータを使用するために研究者を可能にするため感丘再生の標準曲線を確立するために、有毛細胞の切除のためのヴァントランプら 17の手順に従ったが、自分の仕事上に構築された。アッセイはまた、解像度の高いレベルが要求される場合、個々の有毛細胞への分析の拡張を可能にするように設計された。

<table border="0" cellpadding="0" cellspacing="0" width="431">
	<tbody>
		<tr>
			<td>Gentamicin sulfate solution (50mg/ml)</td>
			<td>Sigma Aldrich</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>2 Phenoxyethanol</td>
			<td>Sigma Aldrich</td>
			<td>P1126</td>
			<td></td>
		</tr>
		<tr>
			<td>4-4-diethylaminostryryl-N-methylpyridinium iodide (4-Di-2-Asp)</td>
			<td>Aldrich</td>
			<td>D-3418</td>
			<td>485 nm excitation &#955; and 603 nm emission &#955;</td>
		</tr>
		<tr>
			<td>in methanol</td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Mid Sci</td>
			<td>TP92006</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Matek Corporation</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting&#160; Microscope</td>
			<td>Nikon</td>
			<td>TMZ-1500</td>
			<td>Any dissecting microscope is fine.</td>
		</tr>
		<tr>
			<td>Camera for Imaging</td>
			<td>Nikon</td>
			<td>Q imaging</td>
			<td>Any camera is suitable.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td>NIH Image</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements</td>
			<td>Nikon</td>
			<td></td>
			<td>Any imaging software is suitable.</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Olympus</td>
			<td>FV10i</td>
			<td>Any high resolution fluorescent microscope is suitable</td>
		</tr>
		<tr>
			<td>Aquatic System</td>
			<td>&#160;KG Aquatics ZFS Rack System.</td>
			<td></td>
			<td>Any aquatic system can be used</td>
		</tr>
	</tbody>
</table>

an assay for lateral line regeneration in adult zebrafish

原因人間に聴覚や平衡障害の臨床的重要性、そのようなゼブラフィッシュなどのモデル生物は側線の開発と再生を研究するために使用されてきた。ゼブラフィッシュは、その急速な開発時間と高い再生能力のような研究のために特に魅力的である。現在までに、側線再生のゼブラフィッシュの研究では、主な理由は、これらの段階でneuromasts数の減少の胚および幼虫期の魚を利用してきた。これは横方向のラインの再生/および以前の発達段階での容易な開発の定量分析を行いました。神経学的および非神経疾患の多くのゼブラフィッシュモデルは成魚ではなく、胚/幼虫で研究されているため、アッセイが利用可能になるように、我々はそれが現在にも適用することができる大人のゼブラフィッシュでは、定量的側線回生アッセイの開発に焦点を当て大人のゼブラフィッシュ疾患モデル。ヴァンスペクトラムによるこれまでの研究を踏まえP ら 17アダルトメキシコブラインド洞窟魚やゼブラフィッシュ（ ゼブラフィッシュ ）内有毛細胞の切除のための手順を説明した、私たちのアッセイは、対照群と実験群間の定量的な比較を可能にするために設計されました。これは、横方向のラインの定義された領域内の有毛細胞のゲンタマイシン誘発性壊死を次の24時間の期間にわたって感丘再現の割合（％）に基づいて、回生感丘標準曲線を開発することによって達成された。アッセイはまた、解像度の高いレベルが必要とされる個々の有毛細胞レベルの分析の拡張を可能にするように設計された。

大人のゼブラフィッシュの横のラインの感丘再生を定量化するための手順の最適化。 幼虫のゼブラフィッシュのneuromastsは容易に定量化され;しかし、大人のゼブラフィッシュの側線は6,17,19,20定量分析をより困難ステッチごとneuromastsのはるかに大きい数を持っています。 図1Aに見られるように、ヘッドは、中間部又は尾部のいずれかと比...

Watch this Scientific Journal Video about An Assay for Lateral Line Regeneration in Adult Zebrafish at JoVE.com

An Assay for Lateral Line Regeneration in Adult Zebrafish

神経学的および非神経疾患の多くのゼブラフィッシュモデルは成魚ではなく、胚/幼虫で研究されているため、我々は大人のゼブラフィッシュの疾患モデルに適用することができ、定量的側線再生アッセイを開発しました。アッセイは、1）感丘および2）個々の有毛細胞レベルでの解像度を含んだ。

アダルトゼブラフィッシュ側線再生のためのアッセイ

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

an-assay-for-lateral-line-regeneration-in-adult-zebrafish

Research

JoVE Journal

Biology

12.5K ビュー.  Dr. William M Scholl College of Podiatric Medicine, Rosalind Franklin University of Medicine and Science. 神経学的および非神経疾患の多くのゼブラフィッシュモデルは成魚ではなく、胚/幼虫で研究されているため、我々は大人のゼブラフィッシュの疾患モデルに適用することができ、定量的側線再生アッセイを開発しました。アッセイは、1）感丘および2）個々の有毛細胞レベルでの解像度を含んだ。 

ビデオ: An Assay for Lateral Line Regeneration in Adult Zebrafish

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

アダルトゼブラフィッシュの全腎骨髄の移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

生物学

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

MCF - 7細胞株におけるNanogenomedicineの細胞毒性：MTTアッセイ

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

アダルトゼブラフィッシュにおける眼窩インジェクション

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

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, ...

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 ...

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

アダルトゼブラフィッシュにおける血液検査のための反復採血

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

成体ゼブラフィッシュの生体内心電図検査

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...