我々はpDB600プラスミドの種類のプレゼントのために優れた魚の飼育用さんポーラロイ、そしてスティーブEkkerさんに感謝。これらの研究は、NHGRIからSFへの助成金によって賄われた。

1。選択してテストするための保存された配列のクローニング<ol><li>一つは、最初の機能テストのための潜在的な調節配列を識別する必要があります。我々は、配列を選択する基準として進化的な配列保存性に依存し、最も頻繁にUCSCゲノムブラウザ（http://genome.ucsc.edu/cgi-bin/hgGateway）上のトラックとしてアクセス可能なアルゴリズムのPhastConsを、使用してください。しかし、種を越えて配列保存性を評価するために利用できる他の多くのアルゴリズムがあります。 </li><li>かつて我々のシーケンスを選択している、我々は、ゲノムDNAからそれぞれの配列を増幅するプライマーを設計する。プライマーは、どちらの側に保存された領域に加えて30以上の塩基対を増幅するために選択する必要があります。 </li><li>選択された配列の増幅のために我々は、タカラLA TaqTMシステムを使用してください。他のポリメラーゼは動作しますが、それは増幅中に導入されたエラーの可能性を最小限に抑えるために、校正機能を持つ酵素が含まれているミックスを使用することが重要です。我々は、アガロースゲル電気泳動によりアンプリコンのサイズを確認してください。 </li><li>我々は、Gatewayエントリークローンを生成するために、attL組換え部位を含むエントリーベクターにPCR産物をクローン化するためのPCR 8/GW/TOPO TAクローニングキット（Invitrogen）を使用してください。クローニング反応は、新鮮なPCR産物をより効率的なので、PCRの日の内にクローン作成を実行するのが最善です。 DH5α株の形質転換は、スペクチノマイシン耐性選択に続いて実行されます。 </li><li>我々は、インサートを解放し、アガロースゲル電気泳動によりインサートのサイズを確認するためにEcoRIでプラスミドの〜500ngの消化によってTAクローニングの製品を確認してください。我々は、挿入配列と向きを確認するための順序をお勧めします。増幅に用いたプライマーはまた、シーケンシングに使用できます。 </li><li>エントリークローンから、非コード保存配列は、ゲートウェイLR ClonaseをII酵素ミックス（Invitrogen）を用いて、私たちのゲートウェイ準備デスティネーションベクタ、pGW_cfosGFP1にLR組換えにより往復しています。 DH5α株の形質転換は、アンピシリン耐性の選択に続いて実行されます。 </li><li>我々は、インサートを解放し、アガロースゲル電気泳動によりインサートのサイズを確認するためにEcoRVでプラスミドの〜500ngの消化によってLR組換えの製品を確認してください。正確なクローンが識別されたら、我々は、キアゲンハイスピー​​ドを使用してプラスミドDNA ®プラスミドミディキットを準備する。我々は、さらに、製造業者のプロトコルに従って、QIAクイックPCR精製キットを用いてプラスミドを精製し、30μLのRNaseフリー水でDNAを溶出させる。プラスミドは、ng /μLのと° Cの前の注射に4℃で保存されている125の濃度に希釈する。 </li><li> RNAエンコーディング機能Tol2はトランスポサーゼ酵素はpDB600 plasmid2からin vitroで転写される。我々は、キアゲンミディプレップキットを用いてプラスミドを精製し、次にXbaIで10から20μgの線形化。 mRNAの合成はmMESSAGE mMACHINEキットプロトコール（Ambion）を以下の実施しています。 </li><li>我々は、精製し、キットの指示にしたがってRNAを沈殿させる。我々は、RNaseフリーの水に、単一の反応のための〜1μg/μl、または20μlの最終濃度にRNAを再懸濁し、そしてUV分光光度法によって定量化する。我々はまた、完全長の転写を確認するためにアガロースゲル電気泳動でRNAの〜1μgのを分析する。標準TAEまたはTBEゲルは、この分析には十分ですが、転写キットに付属の変性サンプルバッファーはキットの指示に従って使用する必要があります。 </li><li>我々は、公知のプラスミド（図1）で注射を行うことにより有効性のためにトランスポゼースRNAのそれぞれの新しいバッチをテストします。繰り返し融解と個々のアリコートの再凍結を避けるために、検証した後、RNAを小分けに分割され、-80℃で保存。 <img src="/files/ftp_upload/1722/1722fig1.jpg" alt="図1" /> 図1。ゼブラフィッシュの胚では、コントロールとしてマウスSOX10エンハンサーを注入。（トップ ）明視野像（ 下 ）GFP蛍光像。トランスポゼースRNAのそれぞれの新しいバッチは、有効性について試験される。 </li></ol> 2。モザイクトランスジェニック解析のためのゼブラフィッシュ胚のマイクロインジェクション<ol><li>我々は、無傷chorionsを貫通して、かなりシャープなテーパーとの強力なヒントを得るために設計されたプログラムで、サターP - 97マイクロピペットプラーに1.2 mm外径フィラメントガラスキャピラリーからマイクロインジェクション用の針を引き抜きます。我々は、クリーンなカミソリの刃とマイクロメータのスライドを使用して、約15μm​​の外径に実体顕微鏡下で手でヒントを破る。針は、注射の前に一日行われ、清潔に保つために覆われて保持皿に保存することができます。 </li><li>我々は標準conditions3下で、光の14時間で、定期的な光暗サイクル上で私たちのゼブラフィッシュを維持する。前日に行うmicroinjectionsに、我々は小さな繁殖タンク、ベースタンクで構成されるそれぞれの、マイナスの挿入、およびプラスチックの蓋のタイミング交配のための魚を設定します。私たちは、女性3名またはt当たり2人の男性を配置平行な列にANK。 </li><li> microinjectionsの朝に、ライトサイクルが開始直後に、私たちは産卵のためにクリーンなシステム処理水に雄と雌を組み合わせる開始。それは頻繁に魚をチェックすることが重要である、とも同期バッチを取得するには、産卵の数分以内に卵を集める。産卵は朝を通して魚の新鮮なグループを結合し続けることによって、2〜3時間維持することができます。 </li><li>ラテックス電球を取り付けた&quot;ガラスパスツールピペットを5 1 / 4、広口径で、我々は、部分的に50胚のグループでは、胚Medium2でいっぱい、60 × 15ミリメートルシャーレに採取した胚をソートし、上で収集時間をマーク各皿の蓋。 </li><li>魚が産卵開始したら、我々は氷上でマイクロ遠心チューブに次のように混合することによって、新鮮な注射液を準備します。 <table border="1"><tbody><tr><td>コンポーネント</td><td></td></tr><tr><td>トランスポゾンプラスミド株式（125ng/μl） </td><td> 1μlの</td></tr><tr><td>トランスポゼースRNA株式（175ng/μl） </td><td> 1μlの</td></tr><tr><td>フェノールレッドの株式（H 2 Oで2％） </td><td> 0.5μl </td></tr><tr><td> RNaseフリーの水</td><td> 2.5μl </td></tr></tbody></table> 注射針は、皿を保持するに敷設し、各針のワイド端に注射溶液500 NL降下をピペッティングによって充填されています。液体が毛管作用を介して先端に描画された後、追加のソリューションは、1.5〜2μLの合計に加えることができる。私たちは、それぞれの注射液には、少なくとも2本の針を準備。 </li><li>満たされた針を手にロードされ構成され、メーカーの指示に従って、N2のタンクに接続されて、空気圧ピコポンプまたは類似の圧力インジェクタのニードルホルダーを開催しました。我々は、マイクロスライド上にミネラルオイル中に排出された液滴の直径を測定することにより、注入量を校正する。通常は、20 psiの圧力で120ミリ秒の注入時間は約1 NLの液滴が得られますが、針直径のわずかな変化は、これらのパラメータに影響を与えますし、再校正が針の間に必要となる場合があります。 </li><li>注射は6 - 10Xの倍率で実体顕微鏡下で実行されます。我々は、絨毛膜を通して、1つのセルまたは2セルの段階の早期での胚の卵黄への着実な圧力で注射針を押し、場所に胚を保持するために細かいピンセットを使用してください。理想的には、我々だけで割球の下に卵黄で針の先端に配置します。注射液の約1 NLを追放し、針は撤回される。それぞれのために我々は≥200胚を注入構築する。それはuninjectedコントロールの胚の料理を保存するには、収集された胚の各クラッチのため、また非常に重要です。 </li><li>注射が完了すると我々は、未受精卵、損傷した胚、および失敗した注射（割球の無フェノールレッドを持つ胚を）除去することにより胚をソート。私たちは、その後28.5で胚の培地で胚をインキュベート° Cを観測するための適切な時間まで。 </li></ol> 3。モザイクの発現パターンの解析<ol><li>適切なインキュベーション時間の後、我々は、蛍光用注入胚を調べます。タイミングは、内因性ゼブラフィッシュの遺伝子の正常な発現パターンに依存しますが、我々は通常、最初の5日後に受精を介して毎日見て。 </li><li>我々は、落射蛍光用装備オリンパスMVX10のmacroscopeに蛍光のための胚を調べていますが、低消費電力の撮影が可能な任意の同様の顕微鏡が動作します。初日に死亡または異常に開発された胚の数が多い場合、それらが正常に見えるかどうかを確認するためにそのクラッチ用uninjectedコントロールを見てください。コントロールが正常に見える場合、最も一般的な問題は、注入されたDNAの不十分な純度です。また、初期の段階で胚は、注入されたプラスミドの合計量に敏感であるので、定量が不正確だとあまりにも多くのDNAが注入された可能性があります。 </li><li>胚を調べるとき、彼らはすべての注入構造のためのモザイクになることを覚えておいてください。それは表現の予想されるドメインが小さい場合は特に、胚のすべてにおいて慎重に検討する必要がある。また、式は動的なものであることに注意してください。1日目に強い蛍光が2日目、および式の最初の3日間4日目に何かを見せるかもしれない否定的な胚が消えることがある。 </li><li>我々は、より広範な表現を持ついくつかの胚を選択し、顕微鏡撮影による蛍光のパターンを記録するためにこれらを使用してください。観測の5日後、胚は施設に返却されるべきであり、株式として提起。数ヶ月で、大人の導入遺伝子の生殖細胞系伝達のためにスクリーニングすることができる。 </li></ol> 4。結果我々は広いvを参照してください分析中のエンハンサー要素に応じてパターンと発現のレベルのariety、。我々は通常、非常に組織特異的発現パターンに興味がある、と多くの場合、スケルトンに発現する遺伝子に注目しています。そして多くの場合、スケルトンに発現する遺伝子に注目しています。図2は、我々は軟骨と骨​​での発現を調節するエンハンサーと、私たちの注入胚で観察されているモザイクの発現パターンの例を示します。最後に、テストシーケンスのかなりの部分は、組織特異的発現を調節しないでください。これらの場合、我々は、ほとんどの場合、おそらく非常に少ない蛍光、胚あたり散在セルを参照してください。あまり、我々は、しかし唯一の異所性発現、表皮と骨格筋（図3）の2つの一般的なサイトでは、蛍光表示される場合があります。 <img src="/files/ftp_upload/1722/1722fig2.jpg" alt="図2" /> 図2。注入胚で観察される軟骨と骨の発現パターンの例。 <img src="/files/ftp_upload/1722/1722fig3.jpg" alt="図3" /> 図3。遺伝子と発現調節にエンハンサーの相対的な位置の間にはほとんど相関関係があります。テストシーケンスのかなりの部分は、組織特異的発現を調節しないでください。これらは多くの場合、ほとんど蛍光を持っている、または異所性発現には2つの一般的なサイトがあります：表皮と骨格筋を。 （ トップ ）明視野像（ 下 ）GFP蛍光像。

<ol>
	<li>Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+biological+relevance+of+putative+enhancers+using+Tol2+transposon-mediated+transgenesis+in+zebrafish.">Evaluating the biological relevance of putative enhancers using Tol2 transposon-mediated transgenesis in zebrafish.</a> Nat Protoc. 1 (3), 1297-12 (2006).</li><li>Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genet. 2 (11), e169-169 (2006).</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. , (1995).</li></ol>

我々は、ゼブラフィッシュ遺伝子組み換えを使用して潜在的なエンハンサーの効率的な分析のために我々の研究室で使用されるアプローチを示している。ほとんどの場合、我々は、ヒト遺伝子に関連付けられている組織特異的エンハンサーを探すためにこのアプローチを使用してください。人間と魚の非コード配列との間の時間の最も明白な配列相同性の欠如にもかかわらず、我々はこのア?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Takara LA Taq</td>
<td>&nbsp;</td>
<td>Takara Bio Inc</td>
<td>RR02M</td>
<td>&nbsp;</td>
<tr>
<td>pCR8/GW/TOPO TA Cloning Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K2500-20</td>
<td>&nbsp;</td>
<tr>
<td>Gateway LR Clonase II enzyme Mix</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11791-100</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DH5&alpha;</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>12535-019</td>
<td>&nbsp;</td>
<tr>
<td>Library efficiency competent cells DB3.1</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11782-018</td>
<td>&nbsp;</td>
<tr>
<td>mMESSAGE mMACHINE Kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1340</td>
<td>&nbsp;</td>
<tr>
<td>HiSpeed Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>12643</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick PCR Purification Kit </td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28104</td>
<td>&nbsp;</td>
<tr>
<td>Flaming/Brown Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Pneumatic PicoPump</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>SYS-PV820</td>
<td>&nbsp;</td>		</tbody>
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mosaic zebrafish transgenesis for evaluating enhancer sequences

ヒトゲノム配列の完成は、他の多くの種のそれとともに、非コード配列に特定の機能をascribingの課題を浮き彫りにしました。ゲノムの非コード化割合で実施つの著名な機能は、遺伝子の転写を調節することですが、広く主要なDNA配列からのシス調節エレメントを予測する有効な方法はありません。我々は機能的にはゼブラフィッシュの遺伝子導入を通じて、潜在的なシス調節エレメントを評価するための効率的なプロトコルを開発した。それは空間的、時間的な遺伝子調節に関する情報を提供するため、我々のアプローチは、発達の重要な遺伝子の細胞培養ベースの技術上の重要な利点があります。逆に、それはより速く、より安価なトランスジェニックマウスで同様の実験よりも、我々は日常的に人間のゲノムから単離されたシーケンスに適用します。ここでは、クローニングシーケンスのシーケンスの保全と我々のプロトコルに基づいており、ゼブラフィッシュ胚にそれらをマイクロインジェクションをテストするための要素を選択するために我々のアプローチを示しています。

Watch this Scientific Journal Video about Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences at JoVE.com

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

我々は、分化段階の遺伝子から潜在的なエンハンサー要素を発見し、モザイクのゼブラフィッシュのトランスジェネシスを通じてその機能を評価するために我々のアプローチを示しています。

エンハンサー配列を評価するためのモザイクのゼブラフィッシュのトランスジェネシス

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

mosaic-zebrafish-transgenesis-for-evaluating-enhancer-sequences

Research

JoVE Journal

Biology

13.9K ビュー.  University of Pennsylvania. 我々は、分化段階の遺伝子から潜在的なエンハンサー要素を発見し、モザイクのゼブラフィッシュのトランスジェネシスを通じてその機能を評価するために我々のアプローチを示しています。

ビデオ: Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

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 ...

生物学

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAiは、Necromenic虫にマイクロインジェクションによる遺伝子ノックダウンとトランスジェネシスを媒介 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

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 ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

ゼブラフィッシュ胚骨格筋のレーザー招いた傷害

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

モザイク大人のライブイメージングのためのトマト/ GFP-FLP / FRT方法<em&gt;ショウジョウバエ</em&gt;視細胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Mammalian spermatogenesis is a complex differentiation process that occurs in several stages in the seminiferous tubules of the testes. Currently, there is no reliable cell culture system allowing for spermatogenic differentiation in vitro, and most biological studies of spermatogenic cells require tissue harvest from animal models like the mouse and rat. Because the testis contains numerous cell types - both non-spermatogenic (Leydig, Sertoli, myeloid, and epithelial cells) and spermatogenic (spermatogonia, spermatocytes, round spermatids, condensing spermatids and spermatozoa) - studies of the biological mechanisms involved in spermatogenesis require the isolation and enrichment of these different cell types. The STA-PUT method allows for the separation of a heterogeneous population of cells - in this case, from the testes - through a linear BSA gradient. Individual cell types sediment with different sedimentation velocity according to cell size, and fractions enriched for different cell types can be collected and utilized in further analyses. While the STA-PUT method does not result in highly pure fractions of cell types, e.g. as can be obtained with certain cell sorting methods, it does provide a much higher yield of total cells in each fraction (~1 x 108 cells/spermatogenic cell type from a starting population of 7-8 x 108 cells). This high yield method requires only specialized glassware and can be performed in any cold room or large refrigerator, making it an ideal method for labs that have limited access to specialized equipment like a fluorescence activated cell sorter (FACS) or elutriator.

The STA-PUT method allows for the separation of different populations of spermatogenic cells based on size and density.

STA-PUTの速度沈降を使用して造精細胞型の分離

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

ダイナミックカルシウムの自動分析<sup&gt; 2 +</supイメージシーケンス内の&gt;の信号

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity greatly impacts cell behavior to regulate normal and pathologic processes including cell proliferation, differentiation, adhesion signaling and directional migration. In this regard, the innate ability of certain cell types to migrate towards a stiffer, or less compliant matrix substrate is referred to as durotaxis. This phenomenon plays an important role during embryonic development, wound repair and cancer cell invasion. Here, we describe a straightforward assay to study durotaxis, in vitro, using polydimethylsiloxane (PDMS) substrates. Preparation of the described durotaxis chambers creates a rigidity interface between the relatively soft PDMS gel and a rigid glass coverslip. In the example provided, we have used these durotaxis chambers to demonstrate a role for the cdc42/Rac1 GTPase activating protein, cdGAP, in mechanosensing and durotaxis regulation in human U2OS osteosarcoma cells. This assay is readily adaptable to other cell types and/or knockdown of other proteins of interests to explore their respective roles in mechanosignaling and durotaxis.

A Simplified System for Evaluating Cell Mechanosensing and Durotaxis In Vitro

Many mammalian cells preferentially migrate towards a more rigid matrix or substrate through durotaxis. The goal of this protocol is to provide a simple in vitro system that can be used to study and manipulate cell durotaxis behaviors by incorporating polydimethylsiloxane (PDMS) substrates of defined rigidity, interfacing with glass coverslips.

セルMechanosensingとDurotaxisを評価するための簡略化したシステム<em&gt;インビトロ</em

The composition and mechanical properties of the extracellular matrix are highly variable between tissue types. This connective tissue stroma diversity ...

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 ...