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1。プロトコルのテキスト<ol><li>ゼブラフィッシュの血液を採取する前に、麻酔水を準備する必要があります。 500mlの容量の容器に水槽の水〜200mlを注ぎます。氷のチップ〜200グラムを追加します。温度は、約4℃でなければなりません氷片が溶けたように、それは4℃付近の一定温度を維持するために、より多くの氷のチップを追加する必要があります</li><li>麻酔水の準備が整うと、血液採取のために必要な材料を準備します。 P20ピペッターの低リテンションチップを入れて、それが簡単にアクセスすることができピペッターのままにしておきます。ピペットの先端が汚染の源に連絡することはできません。 </li><li>乾いたガーゼでペトリ皿をカバーしています。鋼の刃とガーゼの別の部分は簡単にアクセスできる場所に配置する必要があります。 </li><li>プラスチックチューブに適合した遠心分離機が必要になります。 </li><li>上記の材料は用意されている場合、麻酔ウィットを最初のゼブラフィッシュを捕獲haの漁網と麻酔のために準備された水の中にそれを解放します。ゼブラフィッシュは、魚に応じて、冷水で3月6日Sは麻酔であることが必要です。それはもはや外部からの刺激に応答しなくなるまで冷水の魚を保管してください。 </li><li>漁網を使用して、ガーゼの尾を切り残して、ガーゼの準備部分に麻酔をかけた魚を置きます。唯一の尾を残して魚の頭と体の上にガーゼを折る。ペトリ皿の上にガーゼで覆われた魚を入れてください。 </li><li>ちょうど臀鰭と尾鰭の間に斜めの切開を作るために鋼の刃を使用しています。血が出てくるが開始されます。この時点では、迅速に作業する必要があります。 </li><li>優しくP20のピペッター（低リテンションチップで事前にロードされた）で出てくる血を熱望する。収集することができる血液の量は、魚の大きさとそれが正常に麻酔をかけたこと程度に依存しています。これは通常、5〜20μlに異なります。血液は、cを停止したとき外oming、優しくチューブに熱望血液を転送します。 </li><li>溶血を避けるために、それが遠心分離機内に配置されるまでの血液チューブは、任意の抜本的な動きをすることなく、非常に慎重に処理することが重要です。 </li><li>溶血を回避するために、血液サンプルは採血後10分以内に遠心分離器で保護されることも重要です。 </li><li>必要に応じて、プールを作り、複数の動物から血液を組み合わせることが可能です。プールされたサンプルは、最初の魚や遠心分離から血液採取の間の遅延が10分を超えない限り、正常に動作します。 </li><li>採血が行われると、0.5グラム（エッペンドルフ遠心機5415D）で10分間、血液を遠心します。 </li><li>遠心分離後、血清は、チューブの最上位層である。ピペットで、よく分割され、安定した両方のレイヤーを維持しながら、単に血清を取得していることを確認し、血清を吸引除去する。 </li><li>新しいマイクロチューブに血清を転送し、それ生化学的な分析に使用できるようになりました。それは生化学的解析を開始するために待っている間に血清を氷中で保存することができます。 </li><li>血清はすぐに使用されません場合は、-18℃で約3ヶ月までのCで凍結することができます。 </li></ol> 2。代表的な結果それは、以前説明したテクニック（ 表2）よりも4倍以上の血液を何を表しているか、各魚から全血5〜20μlを収集することが可能であった。総コレステロール、HDL-コレステロール、LDL-コレステロール、トリグリセリドの生化学的解析は、この手法を用いて採血した後に行われた。男女とも魚の二つのグループが、食物摂取の干渉を避けるために、採血前24時間絶食させた。分析は、総コレステロールおよびトリグリセリドの分析のための小さなスケールの比色試験（LabtestダイアグSA、ブラジル）で行われた、血清中の3μlを使用しました。 LDL-コレステロールとHDL-コレステロール分析は、4μlの血清10μlをそれぞれ用いた。これらの分析は、サンプルあたり10ゼブラフィッシュのプールされたサンプルで実施されました。 血清脂質レベルは、自分の卵とボトムカバー水族館で、2週間の実験期間中に自分の卵へのアクセス権を持っていなかったこと、それらにアクセスした魚の間で比較した。血清分析したところ、血清総コレステロールのレベル（卵362±42 mg / dLのと、卵なし357±13 mg / dL）を、HDL-コレステロール（卵91.22±1.79 mg / dLのと、卵なしで72.14±2.89ミリグラム/ dL以上）、および（卵55.68±10.88 mg / dLのと、卵44.18±9.84 mg / dLのなし）LDL-コレステロールは両群間に有意差は認められなかった。ただし、トリグリセリドのレベルは、対照群に比べ実験群（卵292±64 mg / dLのなし）（P = 0.03卵457±25 mg / dL）を有意に低かった。 <table border="1" ><tbody><tr><td> ＆ニッポン放送P; </td><td> 卵へのアクセス権を持つ </td><td> 卵へのアクセスなし </td></tr><tr><td>総コレステロール（mg / dL）を</td><td> 362.82±73.11 </td><td> 357.69±23.08 </td></tr><tr><td> LDL - コレステロール（mg / dL）を</td><td> 55.69±18.84 </td><td> 44.19±17.05 </td></tr><tr><td> HDL - コレステロール（mg / dL）を</td><td> 91.23±3.11 </td><td> 72.14±5.01 </td></tr><tr><td>トリグリセリド（mg / dL）を</td><td> 457.64±43.78 * </td><td> 292.36±111.28 </td></tr></tbody></table> 表1。両方の研究グループのコレステロールおよびトリグリセリドsericレベル（卵へのアクセス権を持つと卵へのアクセスなし）平均値±標準偏差で表されます。 *統計的に有意（P = 0.03）。 Student t検定。 <table border="1"><tbody><tr><td> AUT<html>オードブル</td><td> 切開の場所 </td><td> 収穫法 </td><td> 麻酔 </td><td> 採取した血液の量 </td></tr><tr><td> Jagadeeswaranら。ら、1999マーサら、2003 </td><td>背びれの後方にマイクロ解剖</td><td>マイクロピペット</td><td>記載されていない 冷たい水でMS222 3パーセント</td><td> 1〜5μL 5 10μL </td></tr><tr><td>イームズら、2010 </td><td>断頭術</td><td>マイクロキャピラリーチューブ</td><td> MS222 0.02％ 28°Cの水</td><td> 5 10μL </td></tr><tr><td>本研究</td><td>臀鰭と尾鰭の間に切開</td><td>マイクロピペットおよび低リテンションのヒント</td><td>水と氷のチップ</td><td> 5 20μlの</td></tr></tbody></table>e_content &quot;&gt; 表2。前述の採血技術と本研究では説明したものとの比較。

<ol>
	<li>Schneider, A. C. R., dos Santo, J. L., Porawski, M., Schaefer, P. G., Maurer, R. L., Matte, U., da Silveira, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implementa&#231;&#227;o+de+um+novo+modelo+de+experimenta&#231;&#227;o+animal+Zebrafish.">Implementa&#231;&#227;o de um novo modelo de experimenta&#231;&#227;o animal Zebrafish.</a> Rev. HCPA. 29, 100-103 (2009).</li><li>Briggs, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish:+a+new+model+organism+for+integrative+physiology.">The zebrafish: a new model organism for integrative physiology.</a> Am. J. Physiol. Regulatory Integrative Comp. Physiol. 282, 3-9 (2002).</li><li>Menke, A. L., Sptsbergen, J. M., Wolterbeek, A. P. M., Woutersen, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+anatomy+and+histology+of+adult+Zebrafish.">Normal anatomy and histology of adult Zebrafish.</a> Toxicologic Pathology. 000, 1-16 (2011).</li><li>Murtha, J. M., Qi, W., Keller, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematologic+and+serum+biochemical+values+for+Zebrafish.">Hematologic and serum biochemical values for Zebrafish.</a> Comp. Med. 53, 37-41 (2003).</li><li>Jagadeeswaran, P., Sheehan, J. P., Craig, F. E., Troyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+zebrafish+thrombocytes.">Identification and characterization of zebrafish thrombocytes.</a> Br. J. Haematol. 107, 731-738 (1999).</li><li>Jagadeeswaran, P., Sheehan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+blood+coagulation+in+the+zebrafish.">Analysis of blood coagulation in the zebrafish.</a> Blood Cells Mol. Dis. 25, 239-249 (1999).</li><li>Eames, S. C., Philipson, L. H., Prince, V. E., Kinkel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sugar+measurement+in+zebrafish+reveals+dynamics+of+glucose+homeostasis.">Blood sugar measurement in zebrafish reveals dynamics of glucose homeostasis.</a> Zebrafish. 7, 205-213 (2010).</li><li>Kilpatrick, E. S., Rumley, A. G., Rumley, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+haemolysis+on+blood+glucose+meter+measurement.">The effect of haemolysis on blood glucose meter measurement.</a> Diabet. Med. 12, 341-343 (1995).</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> J. Vis. Exp. (37), e1717 (2010).</li><li>Kinkel, M. D., Eames, S. C., Philipson, L. H., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+Injection+into+Adult+Zebrafish.">Intraperitoneal Injection into Adult Zebrafish.</a> J. Vis. Exp. (42), e2126 (2010).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+Injection+in+Adult+Zebrafish.">Retro-orbital Injection in Adult Zebrafish.</a> J. Vis. Exp. (34), e1645 (2009).</li></ol>

利害の衝突が宣言されません。

本論文では、ゼブラフィッシュの実験ではさらに、血液や血清分析を可能にする単純な手法を提示します。この手法では、血液パラメータのデータを必要とする将来のゼブラフィッシュ血液の研究に貢献する可能性を秘めています。また、実験モデルとしてのゼブラフィッシュの大きいアプリケーションを許可する必要があります。 この手法は、特別なスキルや正確な?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>ローリテンションのヒント</td><td>アプライドバイオシステムズ</td><td> 022493020 </td><td></td></tr><tr><td>エッペンドルフ遠心機5415D </td><td>エッペンドルフ</td><td>中止</td><td></td></tr></tbody></table>

blood collection for biochemical analysis in adult zebrafish

ゼブラフィッシュは、いくつかのヒト疾患の研究のための動物モデルとして使用されています。それは分子事象の研究と治療戦略のためにだけでなく、いくつかの病態1の生理学的メカニズムを評価するための強力な前臨床試験のプラットフォームとして機能することができます。 研究者は、ゼブラフィッシュ系の探査を可能にするために必要な基本的なテクニックは3欠けていることを推測する可能性がある臓器やシステム2の成人のゼブラフィッシュの生理機能に関連した比較的少数の出版物があります。ゼブラフィッシュの血液生化学値は、最初1999年に初めてJagadeeswaranらによって記述された採血技術を採用したマーサと同僚4で2003年に報告されました。簡単に言うと、血液は背大動脈5の領域では、長さ約0.3センチ、横方向の切開口からマイクロピペットチップを介して収集されました。関与分寸法、これのために熟練した医師を必要とする高精度な技術である。同じ手法は同年6の別のパブリケーションで同じグループによって使用されていました。 2010年、イームズらは、ゼブラフィッシュ7の全血ブドウ糖のレベルを評価した。彼らは、はさみでdecapitationsを実行して、胸の関節にヘパリンマイクロキャピラリーコレクションチューブを挿入することにより、血液へのアクセスを獲得しました。彼らは労働キルパトリックらに基づいて、適切な保存温度で解決された溶血の困難に言及8。我々の研究室でJagadeeswaranのテクニックを使用しようとすると、我々は、コレクションが開始される前に失われる血液かなりの量のを許可しないように、それが正確に適切な場所に切開を行うことが困難であることがわかった。 最近、グプタら9大人ゼブラフィッシュの臓器、もつれらを分析する方法を説明しました。10 intraperitonを実行する方法を説明しましたEAL注射、Pugachら11は、眼窩の注入を実行する方法について説明します。しかし、より多くの作業がより完全にゼブラフィッシュの研究のための基本的な技術を探求するために必要です。 ゼブラフィッシュのサイズが小さい実験モデルとして使用する研究者のための課題を提示します。さらに、規模の小ささ、この与えられた、それは単純な手法が研究者は、ゼブラフィッシュモデルの利点を探求できるようにするために開発されていることが重要です。

Watch this Scientific Journal Video about Blood Collection for Biochemical Analysis in Adult Zebrafish at JoVE.com

Blood Collection for Biochemical Analysis in Adult Zebrafish

本論文では、ゼブラフィッシュの背側大動脈から採血するためのテクニックを紹介します。それはまた、コレステロールとトリグリセリドのレベルを決定するためのテストとして生化学的解析に使用するための血清を得る方法について説明します。

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 ...

blood-collection-for-biochemical-analysis-in-adult-zebrafish

Research

JoVE Journal

Biology

36.1K ビュー.  Centro de Pesquisa Experimental Laboratório de Hepatologia e Gastroenterologia Experimental. 本論文では、ゼブラフィッシュの背側大動脈から採血するためのテクニックを紹介します。それはまた、コレステロールとトリグリセリドのレベルを決定するためのテストとして生化学的解析に使用するための血清を得る方法について説明します。

ビデオ: Blood Collection for Biochemical Analysis 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

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

ゼブラフィッシュの脳室注入

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

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 ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

グリコーゲンの生化学的滴定<em&gt;インビトロ</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

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 ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

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 development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

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 ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

大人のゼブラフィッシュの腎臓におけるネフロン構成と機能の解析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...