著者らは実験室でこのテクニックを開発したローラアンのロウリーを感謝したいと思います。 HSはNIHMH70926によってサポートされています。ジェニファーGutzmanはCSBi /メルクポスドクによってサポートされていました。

プロトコルパート1：マイクロインジェクションのための準備<ol><li>サターインスツルメンツの針プラーを使用してキャピラリー針を引いて、注射の準備。 </li><li>蛍光色素（例えばテキサスレッドデキストランなど）を使用してニードルを埋めます。 </li><li>マイクロマニピュレータ及びマイクロインジェクション装​​置で針をマウントします。 </li><li>斜めの先端を作成するための角度で適切なサイズに針をカット。 </li><li>ドロップサイズを測定し、それがオイルに注入あたり1 2nlの間にあることを確認してください。 </li></ol>パート2：胚の準備<ol><li>興味の段階より、30分前の胚を注入する。例えば、24時間後に受精（HPF）で心室を勉強するために、23.5 HPFで胚を注入する。キンメルらによれば、胚をステージングします。 （4）。 </li><li>実体顕微鏡下で、慎重にピンセットを用いて胚から絨毛膜を除去。 </li><li> 1％アガロースでコーティングされたプラスチック製の皿を使用して、プラスチック製の100から200μlをピペットチップでアガロースの穴を突く。慎重にピンセットを用いて穴からアガロースプラグを取り外します。 </li><li>穴とアガロースの皿に胚のメディアに胚を移す。 </li><li>胚を麻酔し、実験中に動かないようにするためのメディアにtricaineを（ウェスターフィールド（5）にしたがって作られた）を追加します。 </li><li>ゆっくりと穴に胚の尾部を下にして置かないと、射出装置は胚の後方側になるように向きをそれ。 </li></ol>パート3：脳室に注入する<ol><li>脳室に注入するために、針の先端がハイパワーでの胚のような視野の同じフィールドになるようにマイクロマニピュレーションのセットアップを手配する。 </li><li>慎重に下に脳組織を押すことなくr0/r1 hingepointにちょうど後方後脳の細いroofplateを通して針を刺します。 </li><li>だけで十分な染料を完全心室を埋めるために注入する。それは胚の段階に応じて、心室のスペースを埋めるためにいくつかの注射をかかる場合があります。 </li></ol>パート4：イメージング<ol><li>きれいな1％アガロースでコーティングされた皿を使用して、皿に新しい穴を突く、プラグを取り外し、および胚を麻酔し、動きを防ぐために、tricaineを追加。 </li><li>背側のイメージのための穴と位置に心室注入胚の尾部を置きます。 </li><li>透過光を使用してイメージを取る。 </li><li>それは、胚や顕微鏡を移動させないことが非常に重要です。 </li><li>顕微鏡の設定を変更し、蛍光灯を使用してイメージを取る。 </li><li>透過光と蛍光灯の両方で横方向の画像を撮影する胚を再配置。 </li><li> Photoshopで画像処理のためのTIFFファイルとして画像を保存します。 </li></ol>第5部：画像処理<ol><li> Photoshopで画像を処理するために、透過光と同じ位置で撮影した同じ胚の蛍光画像を開きます。すべての設定は、各画像に対して同じであることを確認してください。 </li><li>透過光の像にあなたの蛍光画像をドラッグし、それらを正確に並べる。 </li><li>蛍光画像を選択した状態で、&quot;イメージ、&quot;&quot;調整して、選択&quot;し、&quot;色の置き換え&quot;と白に黒の背景を変更。 </li><li>蛍光画像は、色が均一になるまで&quot;置換色&quot;ウィンドウの右側に&quot;あいまいさ&quot;の要因を調整します。 </li><li> &quot;レイヤー&quot;ウィンドウで、オーバーレイイメージを表示するには、&quot;乗算&quot;を選択します。あなたの脳室空間のイメージが正確に透過光の像を一致させる必要があります。 </li><li>他の画像については、このファイルと繰り返しを保存します。 </li></ol>代表的な結果/成果誤った注入の画像の例はEHに示されている間は、適切な心室の注入イメージは、図1のパネルのADに示されています。適切な注射はシャープなエッジと非拡散色素を持っている。針を心室に遠く挿入されたときに発生すると、以下の脳組織に当たる誤った注射は、、心室スペースの外に目に見えると卵黄にある色素になる可能性があります。 <img src="/files/ftp_upload/1218/1218figure1.png" alt="図1" width="600" height="742" /> 図1。 25 HPF野生型の胚の心室注射。（AD）正しい注射方法と（EH）不正な注入方法は、例として示されています。対応する蛍光像（B、F）と示される正しいと誤った注入法の両方の蛍光と明視野画像（C、G）のオーバーレイと（、E）背明視野像。矢印は、染料が正しくない心室インジェクション（&quot;トラブルシューティング&quot;を参照）に起因する脳室の空間を超えてしまった領域を示している。前方には、すべての画像の左側にあります。 トラブルシューティング： <table border="1"><tbody><tr style="font-weight:bold; font-size:16px;"><td>問題が</tD&gt; <td>原因と</td><td>救済策</td></tr><tr><td>あなたは針で胚を押しつぶしている。 </td><td>あなたの針は、あまりにも鈍さや先端が大きすぎる。 </td><td>新しい針で起動し、それをさかのぼっ損ないません。ベベルは、針注射器の針のようにそれがシャープにする。 </td></tr><tr><td>染料は、胚と卵黄を通してである（図1E - H参照）。 </td><td>あなたの針は、組織を経て心室空間に滞在していない。 </td><td>あなたの針が十分にシャープではないかもしれない、あなたが穿刺の速度を制御することができないので。 </td></tr><tr><td>染料は、前脳であまりにもかすかなかではありません。 </td><td>あなたは、注射のための十分に高い圧力を使用していない、またはあなたの針が十分では、色素を注入するときに脳に到達する前ではなかった。 </td><td>あなたの射出圧力を増加したり、針がわずかに前方に注入中に置きます。より詳細な情報が脳に必要な場合には、中脳後脳境界を通して針を置くことができます。また、単に画像処理の前にさらに拡散するために染料のためのビットを待つことができます。 </td></tr><tr><td>あなたの蛍光画像は、Photoshopで色置換後のエッジの周りに暗いです。 </td><td>あなたは、色の置換]ウィンドウで、&quot;あいまいさ&quot;の設定を変更するのを忘れ。 </td><td> &quot;曖昧さの&quot;カラーの置換]ウィンドウの右端まで移動します。 </td></tr><tr><td>針は、穿刺後脳のroofplateをしません。 </td><td>あなたの針が適切にカットされていません。 </td><td>新しい針を作る。あなたが注射をたくさんやっている場合はそれらが鈍い得ることができるように、いくつかの針が必要になる場合があります。 </td></tr></tbody></table>

<ol>
	<li>Gutzman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> Mech. Dev. 125, 974-983 (2008).</li><li>Lowery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+Classification+of+Zebrafish+Brain+Morphology+Mutants.">Characterization and Classification of Zebrafish Brain Morphology Mutants.</a> Anat. Rec. (Hoboken). 292, 94-106 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> Development. 132, 2057-2067 (2005).</li><li>Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Zebrafish Book: A guide for the laboratory use of zebrafish. , (1995).</li></ol>

このビデオでは、ゼブラフィッシュの脳室の開発に蛍光色素（テキサスレッドデキストラン）を注入する方法を示しています。このメソッドは、周囲の神経上皮とは対照的に、脳室のスペースを視覚化するために使用し、心室空間の形状だけでなく、周囲の脳組織の形状を決定するための非常に便利です。それは、私たちはより良い生きている胎児の時間をかけて脳室の形成と脳の形態形成?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Tubes (needles)</td>
<td>Tools</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>&nbsp;</td>
<tr>
<td>Agarose-Coated dishes</td>
<td>Tools</td>
<td>Agarose (Seakem GTG; Lonza) Dishes (Corning) </td>
<td>Agarose (50027) Dishes (430166) </td>
<td>Make agarose approximately 2-3 mm deep in the dish depending on the stage of embryo you are injecting</td>
</tr>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Zeiss Stereomicroscope Stemi SV 6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Camera with Fluorescence capabilities and with image capture software</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-200 &mu; pipette tips</td>
<td>Tools</td>
<td>Tip One, US Scientific</td>
<td>1111-0806</td>
<td>These are the perfect size to poke the holes in the agarose and have the embryos fit perfectly.</td>
</tr>
<tr>
<td>Photoshop</td>
<td>Image Analysis</td>
<td>Adobe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Loop</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Make these yourself with a glass pipette and hair or fishing line and seal with beeswax.</td>
</tr>
<tr>
<td>Microinjector</td>
<td>Tools</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PL1-100</td>
<td>&nbsp;</td>
<tr>
<td>Needle Puller</td>
<td>Tools</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>Settings (Heat 785, Pull 70, Velocity 40, Time 70).</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tools</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent dye Texas-Red Dextran 70,000MW</td>
<td>Reagent</td>
<td>Molecular Probes</td>
<td>D1830</td>
<td>Other Molecular weights are available if desired.</td>
</tr>
<tr>
<td>Tricaine (3-aminobenzoic acid ethyl ester)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Media</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>According to Westerfield (5)</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish brain ventricle injection

胚の脳の発達の間に適切な脳室の形成は正常な脳機能に必要です。脳室は脳脊髄液で満たされている脳内で高度に保存されて空洞です。それは脳室の形成を生じる流体で満たされる間、ゼブラフィッシュでは、神経管形成後に、神経上皮では狭窄やひだのシリーズを受ける。心室形成の過程、および同時に発生する神経上皮の形状の変化を理解するために、我々は、脳組織に比較して心室の領域を可視化する方法が必要でした。しかし、透明なゼブラフィッシュの胚の性質は、それが困難な心室スペースから組織を区別することができます。したがって、我々は、心室のスペースが蛍光色素を充填し、明視野および蛍光顕微鏡によって撮像される脳室注入の技術を開発した。明視野および蛍光イメージは、処理され、Photoshopで重畳されています。この技法は、明視野像における神経上皮の形状と比較して、蛍光色素による心室空間の可視化が可能になります。ゼブラフィッシュの脳室注入は、初期の幼虫の段階を経て18時間後の受精から採用することができます。我々は、脳室の形成と形態形成の我々の研究だけでなく、脳の形態形成変異体（1-3）を特徴付けるに広くこの技術を使用している。

Watch this Scientific Journal Video about Zebrafish Brain Ventricle Injection at JoVE.com

Zebrafish Brain Ventricle Injection

神経管形成後に、神経上皮は収縮し、真空管が胚の脳室を形成するために胚性脳脊髄液（eCSF）でいっぱいにしながら折る。我々は、よりよい生きている胎児の神経上皮の形状とは対照的に、流体の満たされた空間を視覚化するために、この心室注入技術を開発した。

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 ...

Research

JoVE Journal

Biology

21.2K ビュー.  Whitehead Institute for Biochemical Research. 神経管形成後に、神経上皮は収縮し、真空管が胚の脳室を形成するために胚性脳脊髄液（eCSF）でいっぱいにしながら折る。我々は、よりよい生きている胎児の神経上皮の形状とは対照的に、流体の満たされた空間を視覚化するために、この心室注入技術を開発した。

ビデオ: Zebrafish Brain Ventricle Injection

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

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 ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

キメラゼブラフィッシュ胚における網膜と脳の開発のライブイメージングのためのGFP発現割球の移植

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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, ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

注射とエレクトロポレーションによりゼブラフィッシュ耳胞イントゥMIFモルフォの直接配信は、内耳の開発に影響を与える

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

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 ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

ゼブラフィッシュの幼虫の複数の困難な標的組織をトランスフェクションするために心室インジェクションを用いた遺伝子モルホリノアプローチを逆転

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

ゼブラフィッシュにおける腎毒素マイクロインジェクションは急性腎障害をモデルに

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...