この作品は、KFBの難聴の研究財団からYcをSとNSF（IOS 0930096）への補助金によって支えられている。 KFB：NIH / NINDCD 2 RO1 DC04184、 <a href="https://commons.era.nih.gov/commons/genericStatus.do?actionRole=nonPI&amp;applID=7854446&amp;uhf-token=Ugf7JvudZTCbTUW9jSAR1snMUgg%3D">3R01DC004184 - 08W1</a> 、NSF DBI 0832862、NSF IOS 0930096

1。エレクトロポレーション用電極の作成<ol><li>適当な長さの75ミクロン径のタングステンワイヤーを（AMシステムズ社Carlsborg、ワシントン州）（約3〜5センチ）カット</li><li>モレックスケーブルコネクタ（ピンスタンプ真鍮）を介してタングステンワイヤーを置いて下さい</li><li>ワイヤーに縮小チューブを密封するためにタングステンワイヤと収縮チューブ付きモレックスケーブルコネクタ（SPC技術、シカゴ、IL）をラップし、ブンゼンのバーナーやヒートガンで熱を適用する</li><li>タングステン線の先端をシャープにする、1.0 N水酸化ナトリウムにワイヤーを浸し（コンラッドら、1993）と、他方の電極としてのペーパークリップを使用して、方形波のエレクトロでワイヤーを電解する。我々が使用する条件は、パルス間で50ミリとそれぞれ持続100 msの5ボルトのパルスである。電極には、15〜20ミクロンの直径にシャープされるべきである</li><li>エレクトロポレーションに使用する前に粘土に押された溝内のトレイに配線してください</li></ol> 2。エレクトロステーションの設定<ol><li>テープはマイクロマニピュレータ（世界の精密機器）に75μmのタングステン電極を削った。 </li><li>解剖顕微鏡のステージ（ライカ）の両側に配置マニピュレーター。 </li><li>プロテクトCUY - 21 [編集]矩形波エレクトロ（図1）に電極を接続してください。 </li><li>エレクトロの電源を入れ、電圧、パルス持続時間、及びパルス数を含む、エレクトロポレーションのパラメータを、設定します。この実験のための最も効果的なパラメータは、それぞれがそれぞれのパルス間で100ミリ秒で、5.0ms続いたthree 13ボルトのパルスであることがわかった。エレクトロポレーションは、胚中の気泡を作成する場合、パルスの長さを減らす。 </li></ol> 3。ゼブラフィッシュ耳胞イントゥモルフォのマイクロインジェクション<ol><li>繁殖タンクから収穫のゼブラフィッシュ胚。で胚をインキュベート胚啓発℃で一晩28.5で0.3 PPMメチレンブルー（ウェスター、2005）で培地を。 </li><li>サターP - 97電極プラーとガラス針を引っ張る</li><li> ℃の水浴中で37にアガロースゲル基剤（10mMのペトリ皿に魚の水中の1％アガロース）をウォームアップ</li><li>溶融までの魚の水の熱を1％低融点アガロース（LMPA）と37℃の水浴中で、冷えないように保管してください。 </li><li>モルホリノソリューション（GeneTools、コーバリス、OR）とガラスの針を記入し、適切な直径の先端をカット。マイクロマニピュレータ（クーン）に注射針をマウントします。 </li><li>約10μmの針の先端をカットし、先端がモルホリノソリューションの十分な配信を可能にすることを保証するためにミネラルオイルに注入する。 </li><li> 24時間のpostfertilization（HPF）で胚をDechorinate、0.3 PPMのメチレンブルー魚の水にMS222（tricaine、シグマ）でそれらを麻酔。 </li><li>便利な均一な分析のための右側面を上にしてアガロースゲルベースの​​上に3〜5麻酔胚の位置を合わせます</li><li>それぞれの胚を介して1％LMPAの1〜2滴を入れ、所定の場所に胚を固定するために固めることができます</li><li>耳胞が右側になるようにペトリ皿を回転させる</li><li>耳胞の内腔に針を置き、添付の窒素タンク（図2）でPV820空気圧PicoPump（世界の精密機器）とモルホリノソリューションを注入する</li></ol> 4。エレクトロポレーションの手順<ol><li>すぐに注入した後、エレクトロポレーションステーションに取り付けられた胚を移動する</li><li>耳胞へのちょうど後方組織への正極を置きますが、浸透しない;耳胞にちょうど前脳に負極を挿入する</li><li>フットペダルを使用するか、スイッチを押すことにより電流を適用する。 </li><li>アガロースの魚の水を注ぎ、旋回とガラスピペットでアガロースから胚を削除します。胚を傷つけないように、注意してください。除去が困難な胚の場合は、軽く胚を取り巻くあらゆるLMPAを脱却するために針を使用。 </li><li>場所は、メチレンブルーの魚 ​​の水でプレートの胚および分析のための必要な段階（in situハイブリダイゼーション、etcと微視的anlaysis で 、免疫組織化学的、形態学的）に胚を発生させます。 </li></ol> 5。代表的な結果： <img src="/files/ftp_upload/2466/2466fig1.jpg" alt="図1" /> 図1エレクトロステーション。電極は、シャープ75μmのタングステン線を使用して行われ保護CUY - 21 [編集]矩形波エレクトロにリード線で接続されていました。これらの電極は、マイクロマニピュレータにテープで固定されました。 <img src="/files/ftp_upload/2466/2466fig2.jpg" alt="図2" /> 図2。注入ステーション。すべての胚は、標準化の目的のために右耳に注入した。 <img src="/files/ftp_upload/2466/2466fig3.jpg" alt="図3" /> 図3 3 DPFの胚とアクチンフィラメントのファロイジン染色。 （A）3 DPFの胚injecte制御MOとdは後部黄斑（PM）のファロイジンの強い染色を持っています。 （B）コントロールMOを注入し、エレクトロポレーションされた胚は、注射のみ胚で午後のファロイジン染色の同じようなレベルを持っていた。エレクトロポレーションと一緒にMIF MOの注入は、午後（D）のファロイジン染色の劇的な減少を引き起こした一方（C）単独で耳胞へのMIFのMOとのインジェクションは、ファロイジン染色の低下を引き起こさなかった。 、前黄斑です。 <img src="/files/ftp_upload/2466/2466fig4.jpg" alt="図4" /> 図4 5 DPFの幼虫とアクチンフィラメントのファロイジン染色。標準コントロールモルホリノが（B）に使用されたエレクトロのみインジェクション（）と注入の間に差はなかった。しかし、MIFモルフォとエレクトロポレーションを注入された5 DPFの幼虫は、MIFモルフォ（Cを注入した胚と比べ3 DPFよりもそれほど劇的後黄斑の減少ファロイジン染色（午後）（D）を、、を示した）。 、前黄斑です。 <img src="/files/ftp_upload/2466/2466fig5.jpg" alt="図5" /> 図5 3 DPFにおける胚のアセチル化チューブリン染色。 （A）中の胚は、任意の注射またはエレクトロポレーションを受信しませんでした。 （B）の胚は、エレクトロポレーションがない注射を受信しませんでした。 MIFモルフォと注射とエレクトロポレーション（C、D）の両方を受信する胚は、目に見えて、コントロールと比較してチューブリンの染色を減少していた。 （午後、後部黄斑）。 <img src="/files/ftp_upload/2466/2466fig6.jpg" alt="図6" /> 図6。対照モルホリノ3 DPFにおける胚のアセチル化チューブリン染色。 （A）どんな治療をすることなく胚が後部黄斑（PM）と豊富な神経支配の強力なアセチル化チューブリンの染色を示した。 （B）胚は対照モルホリノを注入。 （C）エンブリオは、エレクトロポレーションで処理した。 （D）制御モルホリノとコントロール胚に注入し、エレクトロポレーションは、治療のコントロールなしに比べて後黄斑と神経支配のアセチル化チューブリンの同じようなレベルがあります。

<ol>
	<li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus.">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> J Neurosci Methods. 50, 123-127 (1993).</li><li>Suzuki, M., Takamura, Y., Maeno, M., Tochinai, S., Iyaguchi, D., Tanaka, I., Nishihira, J., Ishibashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+laevis+macrophage+migration+inhibitory+factor+is+essential+for+axis+formation+and+neural+development.">Xenopus laevis macrophage migration inhibitory factor is essential for axis formation and neural development.</a> J Biol Chem. 279, 21406-21414 (2004).</li><li>Barald, K. F., Kelley, M. W., W, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+placode+to+polarization:+new+tunes+in+inner+ear+development.">From placode to polarization: new tunes in inner ear development.</a> Development. 131, 4119-4135 (2004).</li></ol>

記事の生産は、ビデオとテキストに記載されている試薬を生成する遺伝子のツール、LLCによって後援されています。

私たちは正常に24 HPFのゼブラフィッシュ胚の耳にアンチセンスオリゴヌクレオチドのMOを導入している。注射とエレクトロポレーション後に発生した胚は生存可能であった。胚のエレクトロポレーションを生き延びた場合、それらは通常の治療を受けておらず、胚に比べて速度だけでなく、唯一の注入した胚とのみエレクトロされて​​いた胚に成長した。 我々は、ファロイジンとアセチル化チューブリンで標識を通して耳の注射とエレクトロポレーション後MIFとMIFのような MOの効果を観察した。後黄斑の感覚有毛細胞のアクチンフィラメントのファロイジン染色は、MIFのそのエレクトロポレーションを示しており、唯一のMOを注入した胚と比較した場合、24 HPF段階でMIFのような MOは、有毛細胞および/ ​​または不動の数値の変化を引き起こした（図3）。有毛細胞の数のこの減少は、Shen らの調査結果と整合的である。（提出）MIFとMIFのようにMOSは球形嚢斑における有毛細胞数の減少を引き起こすこと。このエレクトロポレーションを示すMOSとエレクトロポレーションを注入した胚における毛細胞数の減少は、それによって耳胞にのみ注入された胚に比べて形態学的効果の程度を増加させる、細胞膜を横切ってMOを移動するには助けることができる。 statoacoustic神経節の形態の変化は、アセチル化チューブリン染色で観察された。 statoacoustic神経節から神経支配の程度は、神経突起（図5）の分岐減少ショーを注入し、エレクトロポレーションした胚として、影響を受けていた。アセチル化チューブリンの染色が大幅に注入し、エレクトロポレーションした両方の胚に減少するため、また、神経節細胞および/または神経節細胞数の減少の結露をそこに減少させることもできます。 MIFは、神経系の開発（鈴木ら 、2004）ために重要であることが示されているので、エレクトロポレーション後に見られる変化は、MIFとMIFのような神経芽細胞へのMOソリューションの増加トランスフェクションの結果である可能性があります。 直接開発組織へのアンチセンスオリゴモルフォのエレクトロポレーションは、胚に、両方の空間的及び時間的に、その組織の開発中に遺伝子の発現を制御するのに便利なツールです。内耳の組織にMIFとMIFのような MOのエレクトロポレーションは、後黄斑とstatoacoustic神経節の両方の異常形態をもたらした。何の治療を受けておらず、胚を持つかのどちらかのみを注入されているか、あるいは唯一のエレクトロした胚と比較して注入し、エレクトロポレーションされていた胚の後部黄斑の感覚有毛細胞数の減少があった。 statoacoustic神経節から後部感覚パッチの神経支配の程度の低下とSAGの大きさもあった。エレクトロポレーションは、所望の組織で、特定のDNA、RNA、またはMOの主効果を観察し、他の組織上の二次的影響からこれらを区別すること、脳、神経堤とを含むことの利点と、特定の組織に巨大分子をトランスフェクションするための貴重な方法です。内耳の開発に関する耳周囲の間充織（Baraldとケリー、2004）。

<ol><li> MOは：0.3 MIF、MIFのような MO1のための組み合わせのmm、およびMIFのような MO2使用されていた。 5&#39; - acatcggcatgactgcgacagagat - 3&#39;：MIF MOは、ゼブラフィッシュのMIF mRNAの開始コドンに無料です。二つのMOはMIFのように使用したMO1 MIFのような翻訳開始部位に無料です。MO2 MIFのような intron1/exon 2境界でシーケンスASから派生しながら、5&#39; - GTTTCTATATTTATGAACGGCATGA - 3&#39;：5&#39; - GATTCATCCTCTGAAGACGTAAGCC &#39;-3。 5&#39; - CCTCTTACCTCAGTTACAATTTATA - 3&#39;：コントロールMOは、遺伝子のツールから塩基配列をスクランブルされた。 </li><li>魚の水にメチレンブルー（0.3 ppm）の</li><li> MS222（tricaine、シグマ、魚の水中で0.64 mM）を</li><li>低融点アガロース（LMPA、ロシュ） </li><li>フェノールレッド（シグマ） </li><li>抗アセチル化チューブリン（シグマ） </li><li>のAlexa Fluor ® 488ヤギ抗マウス二次抗体（分子プローブ） </li><li>テキサスレッド-ファロイジン（Molecular Probes社） </li></ol>

direct delivery of mif morpholinos into the zebrafish otocyst by injection and electroporation affects inner ear development

近年では、エレクトロポレーションは、眼、脳、およびゼブラフィッシュの体節を含む様々な組織、へのDNA、RNA、およびモルフォの in vivoトランスフェクションのための一般的な手法となっている。遺伝子操作の他の方法に比べてエレクトロポレーションの利点は、特定の組織が電流のアプリケーションによる巨大分子の導入のために、両方の空間的及び時間的に、対象とすることができるということです。ここでは、ゼブラフィッシュの開発内耳の組織にMIFとMIFのようなモルフォをトランスフェクトするためのエレクトロポレーションの使用について説明します。過去の研究では、1から胚に注入MIFモルホリノ- 8細胞期には、広範な形態学的神経系や眼の変化だけでなく、耳をもたらした。開発の後期段階で内耳の組織を標的とすることで、我々は他の組織の影響に起因する二次的効果とは対照的に、発展途上内耳でMIFの主要な効果を決定することができます。後黄斑に関連する神経細胞、神経細胞のプロセス、および毛の細胞の形態を調べるために染色ファロイジンとアセチル化チューブリンを使用することによって、我々はゼブラフィッシュ内耳における標的トランスフェクションのための方法として、エレクトロポレーションの有効性を評価することができた。 24hpf胚の耳小胞は、モルフォとエレクトロポレーションを注射し、その後も治療を受けておらず、または唯一の注入またはエレクトロポレーションされていた胚と比較した。注入とエレクトロポレーションした胚は、有毛細胞数の減少を示したstatoacoustic神経節（SAG）と対照群と比較して少ないSAGのニューロンによる神経支配の減少となりました。我々の結果は、後の段階でotocystsにモルフォの直接配信は、非特異的な神経系1〜8細胞期で配信モルフォの神経堤の影響を回避することを示した。また、脳、神経堤や耳周囲の間充織での主な影響から耳上の二次的な影響を内耳に向けとされていない効果の検討が可能になります。

Watch this Scientific Journal Video about Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development at JoVE.com

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

24hpfでゼブラフィッシュの耳胞に直接モルフォを提供する方法が開発されている。耳胞と浸透をもたらすためにエレクトロポレーションの内腔にモルフォのマイクロインジェクションを使用して、我々は脳にモルフォのエフェクトをバイパスし、内耳に固有の効果を得ることができた。

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 ...

direct-delivery-mif-morpholinos-into-zebrafish-otocyst-injection

Research

JoVE Journal

Biology

14.2K ビュー.  University of Wisconsin, Madison. 24hpfでゼブラフィッシュの耳胞に直接モルフォを提供する方法が開発されている。耳胞と浸透をもたらすためにエレクトロポレーションの内腔にモルフォのマイクロインジェクションを使用して、我々は脳にモルフォのエフェクトをバイパスし、内耳に固有の効果を得ることができた。

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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15マウス胚の子宮内での脳室内注入とエレクトロポレーションで

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

生物学

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

アダルトゼブラフィッシュの内耳感覚上皮のグロスとファイン解剖（ゼブラフィッシュ）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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, ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

dsRNAをインジェクションによるRNAiの干渉にショウジョウバエ胚

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

顔面間葉のエレクトロポレーション

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

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 ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

して、古いニワトリ胚への遺伝子導入<em&gt; EX OVO</em&gt;エレクトロポレーション

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures.
Chitosan-glycerophosphate (CGP) and Ringer&#180;s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT&#160;but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear.
The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR).
Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

A Comparative Study of Drug Delivery Methods Targeted to the Mouse Inner Ear: Bullostomy Versus Transtympanic Injection

Two microsurgery approaches for local drug delivery to the inner ear are described here and compared in terms of impact on hearing parameters, cochlear cytoarchitecture and expression of inflammatory markers.

マウス内耳に標的薬の配布方法の比較研究：Bullostomy<em&gt;対</em&gt; Transtympanicインジェクション

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. ...