この作品は、NIH R01EY020560 - 01でと医学研究賞のWMケックヤングの学者によって賄われていた。著者らは、網膜の調製と注射の撮影中に彼の援助のためにジョセフBedontに感謝します。

1。エレクトロポレーションのためのプラスミド調製エレクトロポレーションに必要なDNAの濃度は5μg/μlです。これは典型的には、所望のプラスミドは、作業量のDNAの精製と濃縮しマキシプレップ（キアゲン社）またはそれと同等のメソッドを使用して増幅する必要があります。次の手順は、作業量のDNAの調製を記載する。 <ol><li>注し100はDNAのμgの（マキシプレップまたは同等の）と操作の容易さのために100μLにボリュームを希釈する。 </li><li>フェノール追加、フェノールの量は約60％のDNA /体積比（μgのDNA：総容積）を得るために計算する必要がありますすなわち：100μgのDNA（100μLで）プラス67μLフェノール（100μgの：167μLを）。充分に混和、これはDNAの過剰なせん断が発生する可能性があるため、上下にピペッティングしていません。 </li><li>室温で14000rpmで5分間、DNAを回転させる。 </li><li>上清を収集し、同じDNAを用いてクロロホルムに追加：ステップ1.2の総体積比、ミックスなど、室温で5分間、14000rpmでステップ1.2とスピンで説明を。 </li><li> DNA溶液の3 M酢酸ナトリウムの10％上清ボリュームを追加し、上清を収集し、穏やかに混合し、2.5 X 100％エタノールの上清ボリュームを追加します。転倒混和します。 DNAは、ミキシングの際に溶液から沈殿してください。 </li><li> 14000rpmで10分間4℃DNA ° Cを回転させる。 </li><li> 70％エタノール350μlですすぎ、4℃で5分間、14000rpmでスピン℃に</li><li>空気乾燥したペレットは、5μg/μLに1 X PBS（細胞生物学のグレード）でDNAを溶かす。これはそれが非常に困難にPBSに溶解するようになるので、サンプルを乾燥し過ぎたしないでください。 </li><li> DNA溶液にファストグリーンの色素（10％株式を）追加し、染料の最終濃度は、注入トレーサーとして機能するように、0.1％です。 </li></ol> 2。 DNAの網膜下注入次の手順は、実体顕微鏡の支援を受けて実行されます。一度慣れた目のオープニング、切開、及び注入のプロセスが回復するために適切に麻酔子犬のための十分な時間ではないが1.5未満分かかります。シャープな30ゲージの針を使用して、慎重に溶融接合上皮（図1C）に沿って切断して目を開く。この基礎となる目の切断に繋がる可能性があるので、無理な力を加えないでください。これは注射を隠すことができます出血につながるとして、まぶたの接合の範囲を超えて切断は避けてください。 <ol><li>これは死亡に繋がる可能性があるので、氷の子犬を埋葬しないで、数分間氷（ 図1A）で新生児マウスを麻酔。氷の上で時間の長さは子犬から子犬の変数である、通常は5分で十分ですが、マウスは、慎重に個人は非常に迅速に対応したり、適切な麻酔を確保するために長い露出が必要かもしれないので監視する必要があります。撤退の反射を確認するために止血と足のピンチを実施する。 </li><li>網膜下腔へのDNAの注入を容易にするために目を開く必要があります。綿棒70％イソプロピルアルコールプレップ（タイコヘルスケア）を注入された二まぶたが（ 図1B）一緒に来て溶融接合上皮を識別する眼。 </li><li>シャープな30ゲージの針を使用して、慎重に溶融接合上皮（ 図1C）に沿って切断して目を開く。この基礎となる目の切断に繋がる可能性があるので、無理な力を加えないでください。これは注射を隠すことができます出血につながるとして、まぶたの接合の範囲を超えて切断は避けてください。 </li><li>平滑末端注射針は、角膜との接合部（ 図1E）の近くに強膜に小切開を作る30 guage針の先端を使用することによって眼の浸透を促進する。これはレンズのパンクに繋がる可能性があるので、あまりにも深く浸透しないでください。 </li><li>各注射（; Exmireマイクロシリンジ、伊藤corpの針外径0.52ミリメートル、内径0.13ミリメートル）のために個別に音量を測定するために注射器の勾配を使用して33ゲージ平滑末端注射針にDNA溶液0.3μlを描く。反対の強膜の壁の抵抗が感じられるまで切開に針を挿入します。針が硝子体チャンバー（ 図2B）を通過するようにレンズを貫通しないように注意してください。 </li><li>ゆっくりとシリンジのプランジャーを押し下げるために人差し指または親指を使って網膜下腔へのDNA溶液を0.3μLを注入する。実験は、DNA溶液を網膜下腔に注入される速度がDNA溶液は、網膜下腔（ 図2C）内に広がる速度を上回るしないようにプランジャーのうつ病の速度を制御するように注意する必要があります。それはこれがDNAを注入されていない、または均等に拡散しない恐れがあるので、針が反対側の強膜の壁にきつく押されていないことが重要であるので、DNA溶液が粘稠になります。成功した注入もになります網膜下腔の部分のDNA溶液の広がり。注入された動物を回転させるときと、緑のトレーサーの基礎となることなく、網膜の領域には明らかであろう。 </li></ol> 3。エレクトロポレーション<ol><li>エレクトロポレーションは、直径10mmのピンセット電極（; BTXインスツルメンツモデル＃522）を使用して実行されます。電極から動物への電気伝導度を最大化し、正極電極と負極に隣接する非注入眼に隣接して充血した目で電極間に注入された子犬の頭を置くためにPBSでピンセット電極を浸す電極（ 図3B）。 </li><li>パルスジェネレータを使用して5矩形パルスを適用する：各パルスは、パルス間の950ミリ秒間隔で80ボルトと持続時間に50ミリ秒です。 </li><li>彼らは氷の麻酔から回復するまで、エレクトロ仔は今まで温めておく必要があります。これは、温暖化のランプの下でまたはスライドウォーマーの上に子犬を置くことによって行うことができます。暖かいスライドを使用している場合は、適切なパッドが金属表面と回復子犬の間に配置されていることを確認してください。リカバリ時に母親にエレクトロポレーション子犬を返します。 </li></ol> 4。分析のための目の準備<ol><li>エレクトロポレーション網膜の収穫と解析の時間は、実験の目的の対象に実行されます。 GFPの発現は3日、エレクトロポレーション後に肉眼的に可視化することができる。 cryoembeddingと以下の手順に従ってセクショニングの。 </li><li>希望のタイムポイントでのエレクトロポレーションの動物を生け贄に捧げる。エレクトロ目をDisectと4で4％パラホルムアルデヒドで固定し° Cを50分間。 </li><li>慎重に強膜、角膜、レンズ、脈絡膜と網膜色素上皮を離れて、マイクロ切開によって眼から網膜を削除します。解剖網膜を著しくエレクトロポレーションの効率を決定するために蛍光で分析することができます。 4℃で一晩30％ショ糖溶液に網膜を転送℃に</li><li> OCT凍結埋め込む-80メディア（サクラファインテック米国）とストアを℃に凍結網膜網膜は、今cryotome上で区分し、ガラススライド上にキャプチャされた可能性があります。 </li></ol> 5。代表的な結果： プラスミドpCAG - EGFPの発現とエレクトロP0新生児網膜の例としては、エレクトロポレーション（P3）と図4のエレクトロポレーション（P14）14日後には、次の3日間で発表されています。エレクトロ網膜組織の面積は、実験から網膜下腔へのDNA溶液の拡散の注入と均一中に発生した組織の損傷の程度に応じて実験の変数です。典型的にはエレクトロポレーション網膜の百分の90から100までは正常に組み込まれ、導入したプラスミドを発現している細胞を特色にする。しかし、エレクトロポレーション網膜の際に組織の形態とエレクトロポレーションの効率を考慮にのみ40〜60パーセントは、比較分析に適したものとなる。針貫通部位のロゼ​​ット形成と網膜剥離はほぼ常に観察され、この領域は、分析には使用しないでください。成功したDNAは、ロゼットと剥離のない針の浸透のサイトへの外側効率的にエレクトロ組織内マイクロインジェクションの結果の間に広がった。グリオーシスはまた、重要な実験的な関心事であり、ほぼ常に針貫通部位に発生します。しかし、ロゼット形成と網膜剥離と同様、グリオーシスは横方向にエレクトロ組織に典型的に重要ではない。このようなグリア線維性酸性タンパク質（GFAP）のような免疫組織化学的マーカーは、分析領域における反応性グリオーシスのレベルが許容されるしきい値内に収まるかどうかを判断するために利用することができる。区分された網膜は、エレクトロポレーション網膜の形態がそのまま残っていると、個々のエレクトロポレーションした細胞が提示されるEGFP発現ラベルことを実証する。 P0エレクトロポレーション（ 図4A - C）とP0エレクトロ（ 図4D - F）14日後には、次の例は3日が表示されます。 P3網膜にエレクトロポレーションした細胞の大部分は、網膜の神経芽細胞層（NBL）に位置しており、網膜の分化神経またはグリア細胞（ 図4B、C）のいずれかの明確な形態的特徴を示すものではありません。 P14によるエレクトロポレーションした細胞は外顆粒層（ONL）と網膜の内顆粒層（INL）で見つけることができます。さらに様々な標識された細胞は、現在INL、光受容体、およびミュラーグリアの介在（ 図4E、F）を含む分化した神経細胞の特徴的な形態学的特徴を表示します。 <img src="/files/ftp_upload/2847/2847fig1.jpg" alt="図1"/> 図1新生児（P0）子犬マウスとDNA溶液の網膜下注入のためのアイの開口部の麻酔。 A）新生児子犬が上に配置麻酔のための砕いた氷のベッド。注入される目は、氷属性に対する防御ダウンに配置されます。 B）アイ開口のため切断されるまぶたの融合接合上皮（B、 矢印 ）の位置を。 C）目を公開するための溶融接合上皮（C、 矢印 ）に沿ってまぶたをカット。 D）溶融接合でのカットは、次の目を開いたが。 E）切開は、平滑末端マイクロインジェクションシリンジを簡単に挿入を容易にするために角膜の下にある強膜の下に目に行われます。スケールバー、B、C、D、E：4ミリメートル。 <img src="/files/ftp_upload/2847/2847fig2.jpg" alt="図2"/> 図2。鈍の挿入は、網膜下腔へのDNAプラスミド溶液のマイクロインジェクションシリンジと注入を終えた。 A）網膜下腔に注射器の先端の目と和解にマイクロインジェクションシリンジの正しい挿入を示す漫画の回路図は。硝子体腔へのDNA溶液の注入、レンズへのソケット、または注射に目を通して注射器の通過は、あらゆるエレクトロポレーションした細胞であれば、いくつかで失敗した実験になります。網膜下腔に注射器の先端部の強膜の壁との和解で行われた切開に注射器のB）浸透。網膜下腔へのDNA溶液のC）注入。スケールバー、B、C：4ミリメートル。次のように略語：R -網膜、L -レンズ、V -硝子、SRS -網膜下腔、BES -鈍は、注射器を終えた。 <img src="/files/ftp_upload/2847/2847fig3.jpg" alt="図3"/> 図3。エレクトロポレーションのためのマウスの上にピンセット電極のオリエンテーション。 A）漫画の回路図は、エレクトロポレーションの目には相対ピンセット電極のプラスとマイナスのパドルの向きを実証。グリーンは、網膜下腔に注入されたDNAの位置を表します。破線の矢印は正極に向かって負に帯電注入DNAの電気泳動の移動を表しています。 DNAの電気泳動では、正極に向かって配向している網膜に負極に隣接して網膜下腔から発生します。 B）ピンセットの正のパドルがDNAに隣接して配置されて目をマイクロインジェクションし、負のパドルは、非注入眼に隣接して配置されます。新生児マウスのピンセットの電極配置のC）高倍率像。破線は、正極に向かってDNAの電気泳動移動方向を表しています。スケールバー、C：5ミリメートル。 <img src="/files/ftp_upload/2847/2847fig4.jpg" alt="図4"/> 図4新生児マウスの網膜（P0）pCAG - EGFPとエレクトロと生後3日目（P3）と生後14日目（P14）で分析した。 AC）P0でpCAG - EGFPとエレクトロ区分P3の網膜の共焦点画像。エレクトロポレーションした細胞の大部分は、網膜神経芽細胞層（NBL）で座っている。 P0でpCAG - EGFPとエレクトロ区分P14網膜の（DF）共焦点画像。エレクトロポレーションした細胞は外顆粒層（ONL）と網膜の内顆粒層（INL）で識別できます。スケールバー、AF：50μmである。

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in vivoでエレクトロポレーションは、DNAの発現プラスミドと網膜細胞の形質転換のための迅速かつ効率的な方法を表します。このメソッドは、実験者が異所的に普遍的に発現プロモーターの制御下に目的の遺伝子を導入することにより、機能研究のゲインを実行したり、関心のある遺伝子を標的とするshRNAコンストラクトを使用して、関数の研究の損失を実行することができます。さら...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td>バッファが飽和フェノール</td><td>インビトロジェン</td><td> 15513-039 </td><td> N / </td></tr><tr><td>クロロホルム</td><td> JTベイカー</td><td> 9180〜03 </td><td> N / </td></tr><tr><td>酢酸ナトリウム</td><td> JTベイカー</td><td> 3470〜05 </td><td> 3 Mの株式</td></tr><tr><td>ファストグリーンFCF </td><td>フィッシャーバイオテック</td><td> BP123 - 10 </td><td> 10％の株式</td></tr><tr><td>イソプロピルアルコール準備</td><td>タイコヘルスケア</td><td> 6918 </td><td> N / </td></tr><tr><td> 30 guage針</td><td>テルモメディカル社</td><td> SG2 - 3013 </td><td> N / </td></tr><tr><td> Exmireマイクロシリンジ</td><td>株式会社イトー</td><td> MS * E05 </td><td> N / </td></tr><tr><td> Tweezertrode（ピンセット電極） </td><td> BTXインストゥルメント、Genetronics株式会社</td><td> 522 </td><td> N / </td></tr><tr><td>電気スクエアPorator（エレクトロ） </td><td> BTXインストゥルメント、Genetronics株式会社</td><td> ECM 830 </td><td> N / </td></tr><tr><td> OCTコンパウンド</td><td>サクラファインテック米国</td><td> 4583 </td><td> N / </td></tr></tbody></table>

in vivo electroporation of developing mouse retina

哺乳類の網膜の開発の過程で発現する遺伝子の機能解析は重要な課題である。関数のノックアウトの構成的または条件付き損失を生成するためにジーンターゲッティングは、コストや手間が、同様に時間のかかる作業のまま。これらの課題に加えて、網膜はノックアウトのアプローチを使用するときに遺伝子が意図しない困惑さにつながる網膜の外側に不可欠な役割を持っていることが表明した。細胞の運命の仕様および/または分化における役割を同定しようとすると、さらに、異所的機能の実験の利得の遺伝子を発現する能力は非常に価値があります。 我々は、エレクトロポレーションにより新生児マウスの網膜へのDNAプラスミドの迅速かつ効率的な組み込みのための方法を提示する。膜1,2,3,4にわたって材料の移転を促進する細胞膜透過性の一過性増加に一定の電界強度の結果、上記の短い電気インパルスのアプリケーション。画期的な作品は、エレクトロポレーションは、脂質二重膜5を介して高度に荷電したDNAの通過を可能にする親水性の原形質膜の細孔の形成を誘導することにより、哺乳動物細胞への遺伝子導入の方法として利用され得ることを実証した。継続的な技術開発は、網膜、ここ6、7、8、9、10に記載されているメソッドを含む複数のマウス組織におけるin vivoでの遺伝子導入のための方法として、エレクトロポレーションの実行可能性をもたらした。 そのDNAが新生児（P0）のマウス及び電気パルスの網膜色素上皮と網膜の間に配置されるようにDNA溶液をピンセット電極を使用して適用される網膜下腔に注入される。マウスの眼の横方向の配置が必要な負極- DNA -網膜-正極配置にピンセット電極を容易に方向することができます。挿入遺伝子の大規模な組み込みと発現は生後2日目（P2）によって識別できます。網膜の細胞の重要な側方移動の不足のため、エレクトロポレーションおよび非エレクトロポレーション領域が生成されます。非エレクトロ地域はどこに適切な内部組織学的コントロールとして機能することができる。 網膜エレクトロポレーションは、CAGのように、ユビキタスプロモーター下に遺伝子を発現する、またはshRNAコンストラクトまたはクレアンリコンビナーゼを用いて遺伝子機能を破壊するために使用することができます。よりターゲットを絞った発現は細胞特異的遺伝子のプロモーターと構造を設計することにより達成することができます。エレクトロポレーションした細胞の可視化は、GFPを発現したり、GFPの発現構築物の共同electroporatingでバイシストロン構文を使用して実現されます。さらに、複数の構成要素は、コンビナトリアル遺伝子の効果や異なる遺伝子の機能の同​​時利得と損失の研究のためにエレクトロポレートされることがあります。網膜エレクトロポレーションは、適切な発現コンストラクトおよび欠失変異体を生成することで、ゲノムのシス調節エレメントの解析に利用することができる。このような実験は、細胞特異的遺伝子発現11の十分なまたは必要なシス調節領域を同定するために使用することができます。潜在的な実験は、構造可用性によって制限されます。

Watch this Scientific Journal Video about In vivo Electroporation of Developing Mouse Retina at JoVE.com

In vivo Electroporation of Developing Mouse Retina

ゲインまたは機能研究の損失のいずれかを実行する目的のためのマウス網膜細胞へのプラスミドDNAの取り込みのための方法<em&gt; in vivoで</em&gt;提示されます。このメソッドは、外部電場の印加によって誘導される細胞の細胞膜の透過性の一過性増加を利用しています。

開発マウス網膜の in vivoエレクトロポレーションで

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

in-vivo-electroporation-of-developing-mouse-retina

Research

JoVE Journal

Neuroscience

In vivo Electroporation of Developing Mouse Retina

20.7K ビュー.  Johns Hopkins School of Medicine. ゲインまたは機能研究の損失のいずれかを実行する目的のためのマウス網膜細胞へのプラスミドDNAの取り込みのための方法 in vivoで提示されます。このメソッドは、外部電場の印加によって誘導される細胞の細胞膜の透過性の一過性増加を利用しています。

ビデオ: In vivo Electroporation of Developing Mouse Retina

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

によるマウス網膜神経節細胞のトランスフェクション in vivoでエレクトロポレーション

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

神経科学

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

の活動の定量化シス</em植片エレクトロポレーションによってマウス網膜で>調節エレメント

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

開発マウス内耳への遺伝子導入による<emインビボで&gt;</em&gt;エレクトロポレーション

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

全体の開発、 ショウジョウバエの脳のex vivoで培養

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

マウスの遺伝子操作は、視床下部を介して開発<em&gt;子宮内</em&gt;エレクトロポレーション

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

血管新生を調査するために新生児マウス網膜の全体のマウント免疫蛍光染色<em生体内で&gt;</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

生体産後エレクトロポレーションおよびマウスの急性脳スライスにおける神経芽細胞移動のタイムラプスイメージング中

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

マウス網膜を使用してCNSにおける血管再生の評価

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...