我々は、ゼブラフィッシュのケアのための縫合、およびR. EthierとL.億年の手助けのためにEC遼に感謝。 AJDは、ハーバード幹細胞研究所、米国腎臓学会、およびNIH / NIDDK（P50DK074030）によってサポートされていました。 CQDは医療発見のためのマサチューセッツ総合病院、基金によってサポートされていました。

1。非トランスジェニックレシピエント魚のコンディショニング。 <ol><li>移植のためのシステムの最大のオスの魚を選択してください。 </li><li>約3分間、またはそれは水泳が、それほど長い間心臓が鼓動を停止することを停止するまでの0.02パーセントTricaine液（pH7）で魚を置きます。 </li><li>ベンチの上に湿らせたペーパータオルで麻酔した魚を置く。 </li><li>魚の腹腔内腔に水の20μlのでゲンタマイシンの40 UGを注入するインスリンの注射器の針を使用してください。腎再生研究のために、このステップでは、強化された移植のための好ましい環境を誘導するために重要である。 </li><li>回復のためにシステムの水に魚を戻す。 </li><li>魚はおとなしいし、振動などの外部刺激に対して非常に敏感かもしれない。回復の3時間後、ガンマ線照射の25グレイで魚を扱う。 </li><li>移植前の食品なしで3日間のシステムに魚を戻す。 </li></ol> 2。遺伝的に蛍光マーカー（EGFPまたはmCherry）で標識前駆ドナー細胞の調製。 <ol><li>直前に外科的処置に、移植されるドナー細胞を準備し、氷上で保管して下さい。 </li><li>腎および造血前駆細胞を含むトランスジェニックライン、：腎研究のために、我々は、Tg（EGFP cdh17）から全体腎臓の骨髄細胞を使用してください。これらの前駆細胞は、遺伝的に標識し、それらが生着し、腎上皮細胞に分化する場合のみ、EGFPを発現するであろう。血管芽細胞の前駆細胞移植のための：造血幹細胞またはTG（EGFP fli1a）のための：というラベルの前駆細胞の他のタイプはまた、TG（EGFP SCL）として、使用することができます。 </li><li> 5分間で0.2％Tricaineにそれらを置くことによって3大人トランスジェニック魚を安楽死させる。 </li><li>腹側に沿って正中切開を作る頭と尾、そして解剖ハサミを削除するには、滅菌カミソリの刃を使用してください。 </li><li>ステレオスコープの下に、ピンセットで浮き袋を含む体腔内の臓器のすべてを削除します。キャビティの背側壁に取り付けられたフラットや色素器官である腎臓を、削除しないように注意してください。 </li><li>次に、背側の壁から腎臓をばらばらにすると2％ウシ胎児血清（1X PBS / 2％FCS）を添加した冷リン酸緩衝生理食塩水の300 ULを含む1.5 mLの遠心管に入れてください。 </li><li>それは、均質化になるまでせん断腎臓に乳棒を使用してください。 </li><li> 50mlのファルコンチューブ内の40μMのセルストレーナーを配置し、バラセルはファルコンチューブに介してフィルタリングできるように、セルストレーナーの上に細胞溶液を移す。 </li><li>優しく汚れをさらにセルを分割するために、ストレーナーに残っている組織の大きな部分を乳棒を使用してください。 </li><li>ファルコンチューブに細胞溶液のままにして、ストレーナーから残りの細胞を収集する1 × PBS / 2％FCS 1.2 mLでストレーナーを洗浄。 </li><li> ° C 300 rcfで5分間4℃で新しい1.5 mlチューブと遠心分離機に細胞溶液を移す。 </li><li> 1X PBS / 2％FCS、1.5 mLで細胞ペレットを洗浄すると粘着性細胞の塊を除外し、再び遠心分離する新しいストレーナを介して渡します。 </li><li>の1X PBS / 2％FCS200μlの細胞ペレットを再懸濁し、PCRチューブに移す。 </li><li> PCRチューブ（4 ° C、300 RCF、1分）遠心し、上清の大部分を取り除く。 </li><li> 2月5日uLの最終容量を取得し、氷上で保存するために、残液中の細胞を再懸濁します。 </li><li>最終的な細胞の収率は腎臓あたり約200万セルにする必要があります。 </li><li>血球計数器で細胞数をカウントし、5 uLのあたり× 10 5細胞の濃度を調整する。 </li></ol> 3。ハミルトンシリンジにセルのソリューションを読み込みます。 <ol><li>滅菌水で3回ハミルトンシリンジをリンス。 </li><li> 3回、エタノール75％とシリンジをリンス。 </li><li> 1X PBS / 2％FCSで3回、再びシリンジをリンス。 </li><li>その後、細胞溶液の1μlのと注射器をロードするだけで注射前に約5 × 10 5個の細胞が含まれています。 </li></ol> 4。直接腎臓に前駆細胞の移植。 <ol><li>魚が移動を停止している約3分、またはまでではなく、あまりにも長い間、心臓が停止することを叩きのための0.02パーセントTricaineでエアコン受信者の魚を麻酔。 </li><li>実体顕微鏡下で、移植側を乾かすために、上、左、腹側に向かってヘッドと紙タオルで魚を置く。 </li><li>その後、乾燥側を公開するために魚をロールオーバー。 </li><li>滅菌ピンセットを使用して、鰓のすぐ後方地域でのスケールを除去。 </li><li>ピンセットで組織を安定化しながらメスで正中線にあるこのエリア1cmの横切開を加えます。 </li><li>深い十分な切開を行うために継続しながら、ピンセットを使用して、組織を公開頭の腎臓を公開する。 </li><li>腎臓の可視化を防ぐ出血の多くがある場合は、魚をeuthenizeと別の受信者の魚を使って最初から作業を開始。 </li><li>頭の腎臓を公開するオープンな組織を覗きながら、直接ヘッドの腎臓に細胞溶液1 ulを注入する。 </li><li>その後、注射針が取り出されると同時に、組織が注入された細胞の漏出を防止するために元の場所に収まるようにピンセットを離します。 </li><li>針のドライバと切開の両側の筋肉を介して貫通することで縫合針を保持する。 </li><li> 2ノットを結ぶと余分な縫合糸を切断。唯一の一針の縫合は、1cmの切開のために必要です。 </li><li>鎮静鎮痛効果で魚を提供するために5時間Tricaineの百万分の10部を含む魚の水中で魚を置きます。 </li><li>通常の栄養補給と回復のためのシステムに戻って魚を置きます。 </li><li>回復の7から18日後、私たちは魚を解剖し、ライブのEGFPの蛍光による腎前駆細胞の生着を​​分析する。 </li></ol> 5。代表的な結果： 移植の全体的な戦略は、図1に示されています。私たちの腎臓研究のために、腎臓は、EGFP蛍光で解剖、分析されます。腎臓前駆細胞の生着と分化は7日間の移植後には早くも検出されます。これは、EGFPの複数のクラスタ+腎上皮細胞（図2）の存在によって明らかである。移植後18日間では、我々は、ドナーの細胞が新しい腎組織（図3）を形成したことを示す、24 EGFP +ネフロンの平均を検出する。長期の生着を示すために、我々は、受信者の魚59日の移植後（図4）を解剖。この魚は腎臓前駆細胞が遊走していることを示唆し、遠隔部位（白矢頭）で、複数のネフロンに加えて、注射の部位（赤矢印）で多くのネフロンを有していた。この手法は、細胞が生着を検出するために約3ヶ月かかる心臓を経由して循環に注入されるときに比べてより速く、より効率的です。 <img src="/files/ftp_upload/2725/2725fig1.jpg" alt="図1"/> 図1。移植。非トランスジェニックレシピエント魚の全体的なスキームは、移植細胞の免疫拒絶反応を抑制するために照射されています。三日後、切開は、魚の側面で行われ、ドナーの前駆細胞が露出ヘッド腎臓に注入されています。切開は、回復のためにシステムに戻されているもの縫合と魚とまで閉じられます。受信者の魚の腎臓は、後の時点で解剖し、生着および前駆活性について分析されています。 <img src="/files/ftp_upload/2725/2725fig2.jpg" alt="図2"/> 図2。七日後の前駆細胞の移植 。腎臓前駆細胞の生着は、EGFP +腎上皮細胞のクラスターの存在（ -拡大図挿入図）によって決定される、7日間の移植後におけるヘッドの腎臓の注入側で検出されている。 <img src="/files/ftp_upload/2725/2725fig3.jpg" alt="図3"/> 図3。 ... 18日後に前駆細胞の移植は、移植後18日後、我々は24 EGFP +ドナー由来のネフロン（ -個々のEGFP +ネフロンの拡大図の挿入）の平均値を検出する。 <img src="/files/ftp_upload/2725/2725fig4.jpg" alt="図4"/> 図4。 59日後の生着と前駆細胞の遊走は。私たちも、59日（赤い矢印の頭）の後に、移植後の長い前駆細胞の生着を検出するために続けた。この後の時点で、ドナー由来のネフロンは、前駆細胞の遊走と一貫性注入（白い矢印の頭）のサイトから離れている部位で検出されています。

<ol>
	<li>Hopkins, C., Li, J., Rae, F., Little, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+options+for+kidney+disease.">Stem cell options for kidney disease.</a> The Journal of Pathology. 217, 265-281 (2009).</li><li>Brittijn, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+lethal+irradiation+in+zebrafish+and+rescue+by+hematopoietic+cell+transplantation.">Effects of lethal irradiation in zebrafish and rescue by hematopoietic cell transplantation.</a> Blood. 104, 1298-1305 (2004).</li></ol>

利害の衝突は宣言されません。

細胞の移植技術は、再生研究のために極めて重要であり、幹細胞活性を測定するためのゴールドスタンダードアッセイです。ここで、我々は、幹細胞や前駆細胞の活性を決定するために直接成体ゼブラフィッシュの腎臓に細胞を移植するための方法を報告する。この手順では、直接腎臓に細胞の注入に続いて、ヘッドの腎臓を露出させる筋肉を通して切開をとることが必要です。切開は、一針の縫合で閉じられ、運営魚は90〜100％生存率を持っています。我々の実験では、移植した腎臓前駆細胞は、7日後の移植によって移植魚80〜100％で検出されています。このテクニックの一つの制限は、切開が容易にメスによって破壊することができる背部大動脈、そばにいることです。しかし、一度マスターして、出血の率は1％未満に減らすことができます。我々の方法は、腎臓に他の細胞型（血液、間質、または血管）だけでなく、小分子を提供するように適合させることができる。この技術の開発は、事前に再生の研究を支援し、in vivoで評価される幹細胞や前駆細胞の可能性を許可します。

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>インスリンの注射針（28ゲージ） </td><td>ベクトンディッキンソン</td><td> 329461 </td></tr><tr><td>セルストレーナー（40 UM） </td><td>ベクトンディッキンソン</td><td> 352340 </td></tr><tr><td> Tricaine </td><td>シグマ</td><td> A5040 </td></tr><tr><td>ハミルトンシリンジ（26ゲージ） </td><td>シグマ</td><td> 20734 </td></tr><tr><td> Ethilon縫合糸（サイズ9から0まで） </td><td>エチコン</td><td> 2819G </td></tr></tbody></table>

transplantation of cells directly into the kidney of adult zebrafish

損傷した組織に幹細胞または前駆細胞の移植に基づいて、再生医療は、慢性疾患1の広い範囲を治療する可能性があります。しかし、ほとんどの臓器は、これらの構造にアクセスするために外科的方法を開発する必要性を必要と、簡単にアクセスできません。このビデオの記事では、我々が直接成体ゼブラフィッシュ、再生および疾患2を研究する人気モデルの腎臓に細胞を移植する方法を説明します。受信者の魚が注入された細胞3の免疫拒絶反応を抑制するために照射によって事前に条件付けされています。私たちは、頭の腎臓が直接臓器への細胞の注入に続いて魚の脇腹の横切開により露出することができる方法を示しています。任意のセルベースの​​再生治療のゴールドスタンダードを - 腎と造血前駆細胞の混合集団を含む蛍光標識全体の腎臓の骨髄細胞を用いて、我々は、ネフロンの前駆細胞が生着し、新たな腎組織に分化できることを示している。この手法は腎臓だけでなく、他の臓器に幹細胞または前駆細胞および/または低分子化合物を精製し、さらに再生医療を研究するために汎用性の高いモデルとしてゼブラフィッシュを向上させる提供に適応させることができます。

Watch this Scientific Journal Video about Transplantation of Cells Directly into the Kidney of Adult Zebrafish at JoVE.com

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

細胞移植は、組織の再生を研究するための、病気の細胞ベースの治療法を開発するために必要不可欠な技術です。ここでは、成人のゼブラフィッシュの魚の腎臓に直接遺伝的に標識された細胞の移植を可能に顕微鏡下手術を示しています。

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 ...

transplantation-of-cells-directly-into-the-kidney-of-adult-zebrafish

Research

JoVE Journal

Biology

14.9K ビュー.  Massachusetts General Hospital. 細胞移植は、組織の再生を研究するための、病気の細胞ベースの治療法を開発するために必要不可欠な技術です。ここでは、成人のゼブラフィッシュの魚の腎臓に直接遺伝的に標識された細胞の移植を可能に顕微鏡下手術を示しています。

ビデオ: Transplantation of Cells Directly into the Kidney of 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

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

糖尿病マウスの腎臓のカプセルに膵島の移植

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

レンズゼブラフィッシュでの移植とアイ変異体の解析への応用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

アダルトゼブラフィッシュからの臓器の解剖

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

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, ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

同系ゼブラフィッシュにおける限界希釈細胞移植を用いた腫瘍増殖細胞の頻度を定量化

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

アダルトゼブラフィッシュ腎臓の解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

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 ...

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 ...