We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

注：全ての実験は、動物管理上のカナダ人評議会のガイドラインに付着し、オタワの動物ケアの倫理委員会の大学によって承認された。 1.手術前の準備と材料<ol><li>プラスミド準備： <ol><li>製造業者のプロトコルに従ってキアゲンMegaPrepを使用してプラスミドを準備します。高いDNA収量を確保し、一晩インキュベートする形質転換された細菌をLBブロスの少なくとも400ミリリットルを接種する。 </li><li>少なくとも5μgの/μlの最終濃度に100〜200μlのTEに再懸濁プラスミド。 -20℃でアリコートのDNAとストア。濃度が低すぎると、析出しないと、これは不純物を追加できるように再懸濁する。新しい準備をやり直す。 </li><li>手術の前に、0.1％希釈されたプラスミド溶液の10または20μLあたり（w / v）のファストグリーン色素の1μlの滅菌水にを2μg/μlに単一のプラスミド（例えばCreを-GFP）を希釈する。 GFPの場合目的の遺伝子をする別個のプラスミド上に同時エレクトロポレーションし、1の比率でGFPプラスミドを、目的の遺伝子を有するプラスミドを注入：GFPでマークされたすべてのセルの両方で同時エレクトロポレーションされる可能性を最大化するために4プラスミド。 </li></ol></li><li>ガラスマイクロキャピラリーチューブを引っ張る - 62℃での一段階のプルを使用して、標準的な針プラー上の（1の外径を以下の針と呼ばれる）。 </li><li>ニードルホルダー、巻かリトラクター、鉗子、手術のためにはさみ、針、ホッチキス、ステープル、ガーゼ、創傷カバー、外科用ドレープ（; 材料表図1A、B）用のはさみ：以下を含む外科パックをオートクレーブ。 </li><li>他の必要なツールを収集します。·エレクトロ、Femtojet Microinjecter、Tweezertrodes（5または7ミリメートル）、生理食塩水、縫合糸、5ミリリットルのシリンジ（ 材料表 ）。 </li></ol> 2.手術のためにマウスを準備<ol><li>疼痛管理のために、時限-Pを注入1時間の手術の前にブプレノルフィン（0.05 mg / kg体重）とregnantマウス。 E17.5の胚の12.5日（E12.5）から任意の場所になりましマウスに、このプロトコルを適用します。 </li><li>バックロードをするmicroloaderピペットチップを使用して、プラスミド溶液で針を引っ張った。これは針が詰まるようになりますように、まだ針の先端に完全に記入しないでください。 </li><li> 1 L /分のO 2で2~5％イソフルランガスを用いてマウスを麻酔し。一度不動、フェイスマスクに配置し、乾燥を防ぐために、目に軟膏を適用する。後ろに1ミリリットルの生理食塩水（0.9％NaCl）を皮下に注入する。 注：イソフルランガスで作業する場合、過剰なガスを奪還し、部屋の中に逃げるのを防ぐためにガススカベンジャーを使用しています。 </li><li>腹部を剃る。 、Chlorohexadineスクラブで腹部を洗って水で洗い流し、そしてchlorohexadine、70％エタノールの最終前処理溶液を適用する。毛皮をカバーするが、腹部曝露（ 図1C、D）を残すために、滅菌ドレープやガーゼでマウスをカバーしています。 </li></ol>子宮内エレクトロポレーションでは、 <ol><li>下の乳首の間に腹部の皮膚の切開を行います。白線に沿って腹腔内に腹膜を切った後、結合組織を引き裂くことによって離れて下層の筋層から肌を引き出します。離れて傷の端を引っ張るために開創器を挿入します。 </li><li>腹腔から子宮を取り出し、（ 図1E）に示すように、それをレイアウト。 <ol><li>別の子犬（例えば、目的の遺伝子とGFP 対 GFPなど）の異なるプラスミドを注射する場合、それぞれの側に仔をカウントし、各プラスミドに何を注入するためにどれと決める。バック暖かく、潤滑胚を維持するために腹部に子宮の片側を交換してください。すぐに（好ましくは、体温に温め）生理食塩水でさらさ子宮を湿ら。 </li><li> （このようなトランスジェニック胚にCreを-GFPを注入する場合など）同じプラスミドを持つすべての仔を注入する場合はONLyが一度に子宮の1ホーンを取り除く。 </li></ol></li><li>注入ワンドのグリップの頭の中でロードされた針を入れて、慎重に小さな外科用ハサミや細かいピンセットで先端をカット。まだ流体が（ 図1Ji-IV）を通過させながら、針はできるだけ長くとどまるように先端をカットします。 <ol><li>足のパドルが押されたときに、0.1〜0.2μLの小さなまだ見えやすい滴が針の先端に表示されるように、マイクロインジェクターで注入（秒）と射出圧力の長さを調整します。別の針の間、可能な限り一貫性のあるこの液滴の体積にしてください。 注：このステップは（議論を参照）胚の生存のために重要である。 </li><li> Femtojetについては、次の注入パラメータを使用します。圧力を注入する（PI）= 250〜300ヘクトパスカル。噴射時間（TI）= 0.8〜1.2秒。補償圧力= 10〜50ヘクトパスカル。必要に応じて、針の間の変動に対応するために、これらのパラメータを調整する。 </li></ol></li><li> S胚を選出し、そっとその背中、（ 図1F）上向きに傾いた頭の上にそれを配置する。 注：これは胚を回すか、シフトが必要な場合があり、あまりにも多くの圧力を適用し、胚を操作しながら羊膜嚢の破裂を避けるためにしないように注意してください。 </li><li>胚が配置されると、矢状洞に三日月形の影のパラレルとして提示心室を、見つける。少し角度心室上記子宮に針の先端をタッチし、子宮壁に針をガイド。必要な圧力の量は、ニードルの鋭さに依存する。心室に皮質に針を押してください。 注：追加の検出可能な抵抗はめったにありませんが、胚の頭は、多くの場合、わずかに押され、その後針が心室に皮質を通過する際に、反跳。 </li><li>プラスミド溶液を注入するために足のパドルポンプ。緑のエリアが表示され、に準拠するまでポンプを継続する心室（ 図1G）の円弧形状。若い胚（E14.5にE12.5）の色素は、多くの場合、対側脳室に拡散する。 注：ポンプの数は、針の先端の直径に依存する胚の年齢（心室の大きさに起因する）、所望の注入量を。胚の間でできるだけ一貫として注入量を保管してください。 </li><li> 3つまたは​​4つの胚を注入する。作業中は生理食塩水で湿ったすべての露出胚を保管してください。 </li><li>バック注入された最初の胚に移動し、正電極は、脳（ 図1H）の所望の領域にプラスミドを描くようにパドルを置く。角度が負極になるよう、電極間の最短経路は、エレクトロポレーションの対象となる構造（ 図2）を介して直接通過する。 </li><li>パドルが所定の位置に入ると、ショックを適用します。胚の年齢およびサイズに応じて、35〜50 Vの5つのパルスを使用する（Tを参照できる1）。 950ミリ秒のパルス間隔で5ミリ秒のパルス長：以下のエレクトロポレーション·パラメータを使用してください。すべての注入された胚を繰り返します。 </li><li>最後の針おそらく乾燥させ、ステップ3.9の間に詰まったとしてステップ3.3のように、新しい事前ロードされた針の先端をカットします。同じプラスミドまたは異なるプラスミドのいずれかで3つまたは​​4つの胚の次のセットを注入した後、ステップ3.9のように衝撃を与え。子宮の片側にあるすべての仔が注入されると、女性の腹部から子宮の第2の面を削除してから、それを暖かく保つために完成した側を交換してください。 </li><li>羊水嚢に外傷を防ぐために、あまりにも多くの圧力を適用するために生理食塩水ではなく、湿った表面を保つように注意して、女性に子宮の両側を返します。単純な連続ステッチで閉じ筋肉層を縫合。ステープル皮膚が閉じ。ステープルが使用できない場合は、簡単な中断されたステッチで皮膚を縫合。 （ 図1I） </li><li> suturiながらNG、徐々に軽減する妊娠中の女性の麻酔を許可するようにイソフルランを断る。 注：手術が完了した後でこれはダムの覚醒を促進します。胚の生存率を改善することが可能な限り短時間麻酔女性を保ち。総手術時間は、女性が最初に麻酔をしたときから開始し、約30分でなければなりません。 </li></ol> 4.アフターケア<ol><li>インキュベーター内または麻酔から回復するための加熱パッド上に縫合し、女性を配置します。清潔な寝具に彼女をベッドと水分補給を維持するケージに標準回復ゲルを配置。術後疼痛管理のために、その後、手術後8時間間隔でブプレノルフィンの雌の3用量（0.05 mg / kg体重）、12時間間隔で4つの追加の用量を与える。 </li></ol> 5.収穫ブレインズ注：脳は成人期にあらゆる年齢投入時に収穫することができる。以下が適用されます出産前に脳を収集するため。子犬が生まれた後、子宮内の順序はリターのすべての子犬のいずれかが同じプラスミドを注射されなければならないので、失われた、またはコントロールおよび実験動物とを区別する別の方法が考案されなければならないことに注意してください。 <ol><li>冷（4℃）を調製し、前の女性安楽死させ、4％パラホルムアルデヒド（PFA）で濾過した。 </li><li>ペントバルビタールナトリウムの致死量（100 mg / kg体重）の腹腔内注射を使用して女性を安楽死させるオープン腹部を切った。子宮の両側を引き出し、子犬が最初に注入したときのようにレイアウトします。 <ol><li>すべての仔がそのようなトランスジェニックごみへのCreを注入する時と同じプラスミドを、受け取った場合には、胚の順序は、したがって、PBS中の女性と場違い子宮をカット、重要ではありません。 </li><li>子犬が異なるプラスミドを注射した場合は、すべての死者子犬に注意して、それぞれの子犬を見つけてください、そしてコントロールとexperimenを注射されたものかを決定女性から子宮を取り出す前に、TALのプラスミド。子犬が子宮をPBS中にいるグループの後に所属を追跡するために注意してください。 </li></ol></li><li> 、個別に各子犬を削除する鋭いハサミやカミソリの刃で首を切る、PBSに頭を置く。トランスジェニックマウスの場合には、遺伝子型決定のための組織サンプルを収集し、そのような12ウェルプレートのように、個別の各ヘッドを維持する。 </li><li>解剖スコープの下では、頭蓋骨から脳を削除皮質ませニックに注意しながら、または正中線を引き裂く。蛍光顕微鏡を使用して、彼らが正常にエレクトロポレーションしたことを示す、脳がGFP +パッチを持っているかどうかを確認してください。 </li><li> 4℃で一晩PFAに浸漬することにより脳を固定してください。 注：PFAは、多くの場合、信号を消光するので、固定前にGFPを発現させるための頭脳を確認してください。 </li><li>凍結前に、少なくとも3日間、20％スクロース中に浸漬を通してCryoprotect脳。 10％、15％および20％のショ糖勾配（各溶液中で24時間）トンを使用O浸透圧に起因する組織損傷を防ぐ。 -80℃で凍結した脳を格納し、10〜14ミクロンの厚さの切片でクリオスタットでカット。 </li></ol> 6.抗GFPとの同時染色 NOTE：抗原回復抗GFPと染色を共同するために必要である場合には、GFPシグナルを損なわない穏やかな抗原回復プロトコルを使用することが重要である。いくつかの抗原回復プロトコルは（さえGFPに対する抗体を用いて）GFP染色と組み合わせて使用​​するにはあまりにも過酷である。前の一次抗体の適用基本クエン酸前処理は、ほとんどのエピトープ24成功した。 <ol><li>のddH 2 O中のクエン酸の0.01 M溶液を作る較正されたpHメーターを用いてpH 6。 </li><li>クエン酸になるまで電子レンジや水浴中でも染色チャンバ内の熱クエン酸75〜80℃に達する。 10〜15分間染色チャンバー内にスライドを入れてください。できるだけ安定した温度を維持する。 </li><li> PBSにスライドを洗う室温で3分間3倍。一次抗体を適用し、標準プロトコルに従って免疫染色を進める。 </li><li> （ 図3A）ノックダウンを確認するために、GFPのための同時標識と、目的の遺伝子によってCreを介在切除を検証します。 </li></ol>

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(44), (2010).</li><li>Yoshida, A., Yamaguchi, Y., Nonomura, K., Kawakami, K., Takahashi, Y., Miura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+expression+of+different+transgenes+in+neurons+and+glia+by+combining+in+utero+electroporation+with+the+Tol2+transposon-mediated+gene+transfer+system.">Simultaneous expression of different transgenes in neurons and glia by combining in utero electroporation with the Tol2 transposon-mediated gene transfer system.</a> Genes Cells. 15 (5), 501-512 (2010).</li><li>Miyagi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sox-2+regulatory+regions+display+their+activities+in+two+distinct+types+of+multipotent+stem+cells.">The Sox-2 regulatory regions display their activities in two distinct types of multipotent stem cells.</a> Mol. Cell. Biol. 24 (10), 4207-4220 .</li><li>Wang, X., Qiu, R., Tsark, W., Lu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+promoter+analysis+in+developing+mouse+brain+and+genetic+labeling+of+young+neurons+by+doublecortin-DsRed-express.">Rapid promoter analysis in developing mouse brain and genetic labeling of young neurons by doublecortin-DsRed-express.</a> J. Neurosci. Res. 85 (16), 3567-3573 (2007).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cereb. Cortex. 19, 120-125 (2009).</li><li>Bouabe, H., Gene Okkenhaug, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=targeting+in+mice:+a+review.">targeting in mice: a review.</a> Methods Mol. Biol. 1064, 315-336 (2013).</li><li>Ferguson, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-autonomous+requirement+for+the+cell+cycle+regulatory+protein,+Rb,+in+neuronal+migration.">A cell-autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration.</a> EMBO J. 24 (24), 4381-4391 (2005).</li><li>Lagace, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+contribution+of+nestin-expressing+stem+cells+to+adult+neurogenesis.">Dynamic contribution of nestin-expressing stem cells to adult neurogenesis.</a> J. Neurosci. 27 (46), 12623-12629 (2007).</li><li>Andrusiak, M. G., Vandenbosch, R., Park, D. S., Slack, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+retinoblastoma+protein+is+essential+for+survival+of+postmitotic+neurons.">The retinoblastoma protein is essential for survival of postmitotic neurons.</a> J. Neurosci. 32 (42), 14809-14814 (2012).</li><li>Shan, B., Chang, C. Y., Jones, D., Lee, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcription+factor+E2F-1+mediates+the+autoregulation+of+RB+gene+expression.">The transcription factor E2F-1 mediates the autoregulation of RB gene expression.</a> Mol. Cell. Biol. 14 (1), 299-309 .</li><li>Ferland, R. J., Cherry, T. J., Preware, P. O., Morrisey, E. E., Walsh, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Characterization of Foxp2 and Foxp1 mRNA and protein in the developing and mature brain. 460 (2), 266-279 (2003).</li></ol>

The authors have nothing to disclose

子宮内エレクトロポレーションで行う際の主要な課題の二つは胚致死性および2）エレクトロ間の変動を減少させることを防止）1である。鈍い針が胚にダメージを与えるような致死性を防止する上で最も重要な要因の一つは、針の品質である。針を切断する場合、まだ足のパドル（図1J）のポンプ以下の表示される流体の0.1〜0.2μlと見える小滴を可能にしながら、できるだけ長く先端を保つ。針があまりにも鈍いですまたはシャフト短すぎる場合は、結果として組織の損傷は、胚を殺すことができる。針の先端が細かすぎる場合は逆に、十分なプラスミド量を注入するために時間がかかりすぎる。研究者の手の避けられない振動や胚のわずかな回転のために長すぎるの原因の損傷のための脳内に針を残す。さらに、若いに注射を可能にするために、鋭い、面取りヒント針を作るためにmicrobevelerを使用、より脆弱な胚、これは胚のE13.5及び高齢のために必要であってはならないが。 妊娠中の女性のストレスレベルは、胚の生存に影響を与える別の重要な要因である。ダムのストレスレベルが高いほど、より多くの可能性が高い彼女はゴミを中止します。この理由から、麻酔時間を最小限にし、前と手術後できるだけハウジングを変化させることが重要である。バックグラウンド系統は、ストレスレベルに影響を与えることができ、調査中の表現型が異なるバックグラウンド系統間で維持されると仮定すると、そのようなCD1などの歪みへの不安になりやすい異系交配コロニーに必要であり得る。 IUEに唯一最大の制限は、エレクトロポレーションの間に固有の変動である。各エレクトロポ脳による注入量、パドルの配置、パドル接触胚時代の変化に異なっている。針が可変であるので、一定量を注入することができない - むしろinvestigatorは、すべての胚と同様の金額をお届けするために緑のパッチのサイズを概算しなけ​​ればならない。胚は、流体充填された嚢に浮いているため、同様に、定位注射のために行われているように、体系的に、胚の頭部の上のパドルの位置及び姿勢を計測することは不可能である。最後に、胚の年齢のわずかな違いは、実験結果に重大な影響を与え得る。このため、同腹仔の間の比較をお勧めします。

ラジアル移行が早い皮質の開発の中心的なプロセスです。このような正確な有糸分裂の終了と神経分化、細胞極性、細胞骨格の動態および膜貫通受容体の発現の調節、ならびにそのような放射状グリア足場の形成のような非細胞自律的因子などの細胞自律的な様々な要因に依存し渡り鳥ガイダンス分子1-3の分泌。これらのメカニズムのいずれかの破壊がトランスジェニックマウスモデル4-8にニューロン移動を損なうことができるので、マイグレーションの欠陥の根本原因を決定することは、複雑で困難なプロセスであることができる。 子宮内エレクトロポレーション（IUE） で補完し、簡素化するために使用することができるそれによって、遺伝子ノックアウトモデルの表現型との解釈は、放射状の移行7,9-11に必要な重要なメカニズムを解明。 子宮内エレクトロポレーションでは （IUE）はによるプラスミドcarryiプロセスであり、<html>目的の遺伝子およびレポーターの両方をngのは、マウスまたはラット胚の脳内脳室に注入した後、電流12〜14の使用を介して心室の内側を覆う細胞に引き込まれる。これは研究者がアップまたは電気穿孔ニューロンの発生または機能の目的の遺伝子のダウンレギュレーションの効果を分析することができます。 IUE後、脳を免疫組織化学7,9-11、電気生理学15または細胞培養物16,17のために処理することができる。 IUEの主な利点は、遺伝子発現の高度に特異的な操作が可能である。さらに、IUE有向電流（ 図2）を介して発達中の脳の特定の領域を標的化するために使用することができる。 IUEも10,18-21（テトラサイクリン誘導性遺伝子発現が一例である）は、種々のプロモーターまたはプラスミド活性化システムの制御下で遺伝子を担持するプラスミドの注入を介して特定の細胞型を標的化するために使用することができる。 jove_contentは&quot;&gt; IUEのCreのLox仲介遺伝子の切り出しシステム7-9,11,22と組み合わせて使用することができる。Creタンパク質のためのメッセージをコードする遺伝子を含むプラスミド（フロックス対立遺伝子についてホモ接合性の胚にエレクトロポレーションすることができる遺伝子または特異的なDNA領域）のCre媒介DNA組換えのための2つのloxP部位によって隣接される。Creがその後のフロックスallele.The効果のノックアウトを生成する、特にエレクトロポ神経前駆細胞に目的の遺伝子の組換えを誘導する神経遊走および個々のニューロン内の開発にノックダウンタンパク質は、次に検討することができる。Creリコンビナーゼのエレクトロポレーション、無傷の支援環境を残して、影響を受けた細胞のわずかな集団において組換えを誘導する。これとは対照的に、特定の細胞型の制御下のCreの組織特異的発現プロモーターは、全体の組織全体で発生した両方の移行神経芽細胞と周囲の環境が影響を受ける可能性があること。したがって、JUXので、これらの2つのアプローチのtapositionは、与えられた移行欠陥が自律的または細胞非自律的なメカニズムをセルに起因しているかどうかを判断できます。実験的なシステムの両方の不良移行は細胞自律的メカニズムから観察された表現型の結果を示唆している。組織特異的Creリコンビナーゼモデルにおける欠陥の移行とのCreのエレクトロポレーション後の通常の移行は、目的の遺伝子は、非細胞自律的な機構を介して作用していることを示している。 IUEもノックアウト動物を7,9,10に潜在的に相互作用する遺伝子をエレクトロポレーションによって、レスキュー実験を行うために使用することができる。例えば、研究者は下流のターゲットを電気穿孔すると、移行欠陥が電気穿孔ニューロンで修正されているかどうかを決定することによってトランスジェニックモデルにおける移行の表現型を救出しようとする可能性があり。これが成功したレスキューが通常移行がSPEの中でタンパク質の発現を操作することによって復元することができることを示している付加的な利点を有しているcificニューロン、周囲の環境は依然として標的遺伝子を欠損であっても。再び、このアプローチは、欠陥のあるメカニズムは、細胞自律的であるかどうかを決定するという利点を、トランスジェニック系統を交配または生成するより効果的な、より多くの時間とコストである。 IUEはノックアウト胚（ 図4C）にレポータープラスミドの電気穿孔を通って移動するニューロンを追跡するために使用することができる。手術時に心室の内側を覆うだけのニューロンが17エレクトロポレーションされると、IUEは、 インビボでのそれらの形態の視覚化の利点を有する特定の時間に生まれたニューロンを追跡するために使用することができる。 最後に、このような内側または背側終脳、海馬または神経節隆起（ 図2）のような脳の領域を標的とするIUEを使用することが可能である。これは、研究者の合併症の結果の特定の領域とは独立に遺伝子機能を調査するために力を与える近隣の構造にノックダウンタンパク質からる。 網膜芽細胞腫タンパク質（pR​​bの）のp107およびp130のポケットタンパク質ファミリーを含んでよく、細胞周期の出口のレギュレータを設置している。しかしながら、これらのタンパク質はまた、神経発生の細胞周期の独立した側面を調節するという証拠が増えている。我々は以前に示したように、pRbのとP107は、接線4,23とラジアル移行6の両方で重要な役割を果たしている。ここでは、トランスジェニックモデルでのCreの子宮内エレクトロポレーションでの使用を例示するために、神経遊走6にポケットタンパク質ファミリーのメンバーのpRbおよびp107の役割を実証する。要約すると、IUEは遺伝子欠失（または過剰発現）の細胞自律的な効果を分析する強力な方法を提供します。モデルをノックアウト特定の組織と組み合わせると、IUEは、神経移行を制御するメカニズムに関する追加情報を提供することができます。

<table><tbody><tr><td>ECM 830 Square Wave Electroporator</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td>Electroporator only &amp;ndash; buy cables separately (45-0204)</td></tr><tr><td>1250FS Footswitch for ECM 830</td><td>BTX Harvard Apparatus</td><td>45-0211</td><td>Foot paddle is necessary for hands-free electroporation</td></tr><tr><td>Tweezertrodes</td><td>BTX Harvard Apparatus</td><td>45-0489 (5mm)&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 45-0488 (7mm)</td><td>Platinum forcep style electrodes</td></tr><tr><td>Femtojet</td><td>Eppendorf</td><td>920010504</td><td>Microinjector</td></tr><tr><td>Griphead 0</td><td>Eppendorf</td><td>920007414</td><td>Holds the pulled needle</td></tr><tr><td>Universal Capillary Holder</td><td>Eppendorf</td><td>920007392</td><td>Connects griphead to tube</td></tr><tr><td>Injection Tube</td><td>Eppendorf</td><td>920007431</td><td>Connects capillary holder to Femtojet</td></tr><tr><td>Foot Control</td><td>Eppendorf</td><td>920005098</td><td>Foot paddle for hands-free injection</td></tr><tr><td>Microloader Tips</td><td>Eppendorf</td><td>930001007</td><td>Tips for loading the pulled capillary tubes</td></tr><tr><td>Plasmid Mega Kit</td><td>Qiagen</td><td>12181</td><td>5 QIAGEN-tip 2500, Reagents, Buffers</td></tr><tr><td>Fast Green FCF</td><td>Sigma-Aldrich</td><td>F7252-5G</td><td>Non-toxic dye</td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td>For trimming the needles</td></tr><tr><td>Bonn Strabismus Scissors</td><td>Fine Science Tools</td><td>14084-09</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11050-10</td><td>Straight</td></tr><tr><td>Colibri Retractors</td><td>Fine Science Tools</td><td>17000-03</td><td></td></tr><tr><td>Stapler</td><td>Fine Science Tools</td><td>12031-07</td><td>For 7mm clips</td></tr><tr><td>Castroviejo Needle Holder</td><td>Medical Tools</td><td>SNH-6737</td><td>Straight, with lock</td></tr><tr><td>Needle Puller</td><td>Narishige</td><td>PC-10</td><td>For pulling microcappillaries</td></tr><tr><td>Microcapillaries</td><td>Drummond</td><td>1-000-800</td><td>0.4 mm inner diameter glass capillary tubes</td></tr></tbody></table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

トランスジェニックマウス系統でのCre-Lox系を用いた遺伝子欠失がタンパク質の機能を研究するために使用される強力なツールである。しかし、非常に特異的にCreモデルを除いて、組織または細胞集団全体のタンパク質の欠失は、しばしば複数の相互作用のメカニズムに起因する複雑な表現型をもたらす。問題になっている、またはそのセルの環境の障害から生じる非細胞自律的なメカニズム、から細胞に固有のものである細胞自律的な機構の混乱から表現型の結果は、識別することは困難であるかどうかを決定する。 in vivoでのコンテキストでタンパク質機能への洞察を得るために、 子宮内エレクトロポレーション（IUE） で開発皮質またはいくつかの他の選択された脳領域内の細胞の小さなサブセットで遺伝子欠失を可能にします。 IUE背側終脳、内側終脳、海馬、または神経節隆起を含む、特定の脳領域を標的とするために使用することができる。これは、観察oをを容易に健全な環境の文脈における細胞自律的な遺伝子欠失の結果、F。このプロトコルの目的は、IUEが細胞自律的タンパク質欠失の非細胞自律的効果とを区別するに重点を置いて、フロックスジェニックマウス系統におけるラジアル移行の欠陥を分析するために使用することができる方法を示すことである。制限された細胞集団におけるIUE媒介遺伝子欠失に対する全体皮質内の遺伝子欠失から生じる表現型と比較することにより、脳の発達におけるタンパク質機能へのより深い洞察は、分離のいずれかの技術を使用しても得ることができる。

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (Figure 2A), place the positive electrode on top of the green crescent, with a slight rostral angle. For the medial cortex (Figure 2B), switch the orientation so that the positive electrode is over the contralateral non-injected ventricle, this time with an even larger forward angle. To target the hippocampus (Figure 2C), place the positive electrode over the contralateral side, but instead angle the positive electrode caudally (electroporations must be done at an age when the neural precursors are still part of the cortical hem, prior to migration to the final location of the hippocampus). To target the ganglionic eminence (Figure 2D) place the paddles on the embryo in line with the ears, with the positive electrode slightly lower than the negative electrode. Representative images of these electroporated regions are shown in Figure 2.
It is important to validate Cre-mediated excision in electroporated brains. The ideal approach is to perform in situ hybridization or immunohistochemistry against the transcript or protein in question. Unfortunately, as is the case for pRb, reliable probes or antibodies are not always available against the deleted gene. In these cases, alternative approaches, such as staining for a downstream target regulated by the deleted gene (Figure 3A) or confirming excision of the targeted allele in electroporated sections by PCR (Figure 3B), can be used. For example, Figure 3A confirms Cre mediated excision by reproducing a well-documented effect of pRb/p107/p130 gene deletion in electroporated neurons. Following electroporation of pRb f/f; p107 -/-; p130 f/f neurons with the pCig2-Cre plasmid, these triple knock out GFP+ cells continue to express the cell cycle progression marker Ki67, which is a known effect following excision of pocket proteins25. Control GFP+ neurons electroporated with the empty pCig2 plasmid do not express Ki67. The Cre plasmid should also be tested in vitro through co-transfection with a plasmid carrying a reporter gene behind a floxed stop codon, such as the NL-dsRed plasmid (Figure 3C) in a cell line, but this should only be used in combination with other in vivo techniques.
Due to the variation between electroporated brains, analysis of electroporated neurons requires calculating the percentage of total GFP+ neurons that portray a specific characteristic, such as expression of a protein marker or location within a given cortical layer. It is crucial to compare electroporated sections from similar anatomical locations within the brain relative to known landmarks. For example, images for Figure 4 were taken at the level of the anterior commissure. Furthermore, due to horizontal migration, images taken near the edge of the electroporated regions have a higher proportion of mature neurons that have reached the cortex, while images taken near the middle of the electroporated region have both mature and immature GFP+ cells. To insure consistency, it is important to quantify images selected from similar places within the electroporated patch.
Following Emx1-Cre mediated deletion of pRb and p107 in pyramidal neurons in the dorsal telencephalon, a population of neurons expressing the upper cortical layer marker FoxP1 were mislocalized to the intermediate zone (IZ) (Figure 4A). There are significantly more FoxP1+ cells in the IZ of double knock out (DKO) brains compared to control brains with a corresponding decrease in the number of FoxP1+ cells in the upper cortical layers (above layer IV) of DKO brains than found in the upper layers of control brains (Figure 4B). There was no corresponding defect in the radial glial scaffold (Figure S2 of Svoboda et al., 2012) 6. Here we compare expression of cortical layer markers Tbr1, FoxP1 and Cux1 in pRb f/+; p017 +/-; Emx1-Cre + (wild type (WT)) brains to expression of these same markers pRb f/f; p107 -/-; Emx1-Cre + (DKO) brains. Heterozygote embryos can be used as controls since pocket proteins exhibit autoregulation at the protein level and heterozygote animals are aphenotypic 26.
It was unclear whether these FoxP1+ cells were found in the IZ because these cells were immature neurons inappropriately expressing FoxP1, or whether they were earlier born neurons that had failed to migrate. To distinguish between these possibilities, we electroporated GFP into the wild type (WT) and double knock out (DKO) embryos at E14.5 and tracked the progress of these neurons over 4 days. Although this technique does not conclusively identify the final cell division, as with BrdU birthdating, it does allow the tracking of newborn neurons since it marks the discrete set of neurons derived from cells lining the ventricle at the time of electroporation 17. As shown in Figure 4C, all of the GFP+ neurons reach the uppermost cortical layer in the WT brains, but only a subset of these neurons reach the top of the cortex in DKO brains, indicating that there is in fact a defect in the migration of upper layer neurons, rather than improper expression of neuronal markers (similar results were obtained through traditional BrdU birthdating 6).
Neuronal migration is a highly complex process that is regulated by a multitude of interconnected mechanisms 1-3. As it is very difficult to identify the mechanism underlying a migration defect in an uncharacterized transgenic model, distinguishing cell autonomous versus non-cell autonomous defects can facilitate phenotype characterization. If following electroporation of Cre-GFP, GFP+ neurons fail to migrate in an otherwise wild type brain, this indicates that the mechanism disrupted by gene deletion is specific to the electroporated cell. Alternatively, if the electroporated neurons migrate normally in the wild type brain, this suggests that the cause of aberrant migration in the transgenic embryo is due to an environmental defect. Following electroporation of Cre-GFP in control pRb f/+; p107 +/- and double knock out pRb f/f; p107 -/- embryos, GFP+ neurons were found to migrate into the cortex in pRb f/f; p107 -/- brains, but many of these neurons failed reach the upper cortical layers as they did in control brains (Figure 4D) 6. There were significantly lower percentage of GFP+ neurons within the layer III/IV, marked by expression of FoxP127, in pRbf/f; p107 -/- electroporated brains than in control brains (Figure 4E). A Cre-GFP fusion protein was used to limit GFP fluorescence to the cell body and facilitate localization of the neuron to a specific cortical layer. Since the phenotype following electroporation of Cre is less severe than the phenotype following gene deletion throughout the cortex, this suggests that the original phenotype shown in Figure 4A is a combination of multiple mechanisms, both cell autonomous and not cell autonomous. Alternatively, it is possible that the time delay following electroporation, during which Cre is expressed, DNA recombination is performed, pRb is downregulated and the effects are manifested, is long enough to allow the electroporated neurons to reach the cortex before a migration defect becomes apparent.
It should be noted that the distinction between cell autonomous and non-cell autonomous mechanisms should be interpreted with caution in brains with high electroporation efficiencies, where 100-200 or more neurons are electroporated in a single 14 &#181;m thick section.If a high enough proportion of neurons are electroporated, GFP+ neurons may influence each other and create a small microenvironment. To be truly cell autonomous, migration defects must be detected even when electroporation efficiency is very low (10-30 GFP+ neurons per section). If necessary, the number of transfected cells can be lowered by decreasing voltage, pulse number or DNA concentration. All of these factors must be taken into consideration when analyzing the results following electroporation of Cre in a transgenic model.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig1highres.jpg" width="475" /> 
Figure 1: Performing In utero electroporation. (A) Sterile surgical set up. (B) Surgical tools from left: fine scissors for cutting needle tips; stapler; forceps; wound retractor; surgical scissors; needle holder. (C) Prepped pregnant mouse with shaved and disinfected abdomen. (D) Mouse covered with sterile drape prior to incision. (E) Uterus with E13.5 embryos removed from abdominal cavity. (F) Injecting pups in the ventricle. (G) Close up of injected pup; note the blue crescent on the embryo&#8217;s head indicating that the ventricle was successfully injected with the plasmid solution (arrow). (H) Paddles are placed to electroporate the lateral cortex. (I) Mouse post-surgery. (J) Preparing sharp needles is a crucial part of in utero electroporation. Examples of uncut needles (i, iv), good needles (ii, v) and poor needles (iii, vi) are shown at low (i-iii) and high (iv-vi) magnification. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig2highres.jpg" width="600" /> 
Figure 2: Targeting different brain regions. Diagrams A-D show paddle placement for targeting different regions of the forebrains. The dark blue circle represents the paddle in front of the embryo while the light blue circle represents the paddle behind the embryo. Positive and negative electrodes are indicated with the appropriate sign. (A) Electrodes angled to target the lateral cortex. (A&#8217;, A&#8221;) Example of electroporation of the lateral cortex, three days after surgery. (B) Electrodes angled to target the medial cortex. (B&#8217;, B&#8221;) Example of electroporation of the medial cortex. (C) Electrodes angled to target the hippocampus. (C&#8217;, C&#8221;) Example of electroporation of the hippocampus. (D) Electrodes angled to target the ganglionic eminence (GE). (D&#8217;, D&#8221;) Example of electroporation of the GE. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig3highres.jpg" width="600" /> 
Figure 3: Confirming Cre-mediated gene excision in electroporated neurons. (A) Cre mediated excision can be confirmed by co-staining for a downstream target of the deleted gene. pRb f/f; p107 -/-; p130 f/f embryos were electroporated with either the empty pCig2 plasmid or the pCig2-Cre plasmid and stained for GFP and Ki67, a marker of cell cycle entry. Double stained cells were seen in neurons electroporated with Cre (arrows in inset) but not in neurons electroporated with pCig2 alone (arrowheads). (B) Excision of pRb was also confirmed by performing PCR with genotyping primers on DNA extracted from the tissue sections removed from the slides. Sections were divided down the midline and the electroporated side of the section was pooled. The upper band represents the floxed allele and the lower band represents the wild type allele. The arrow indicates the excised allele in the electroporated side of the brain only. Because pRb f/+; p107 +/- heterozygotes were used as controls, an excised allele can be seen in the electroporated side of the brain in both the control and knockout brains. (C) Cre-mediated excision can also be confirmed in vitro using plasmids with floxed stop codons upstream of RFP, such as NL-dsRed. RFP is only expressed in HEK cells when co-transfected with Cre-GFP in Hek293 cells (bottom row), indicating that excision was successful. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/51983/51983fig4highres.jpg" width="600" /> 
Figure 4: Using IUE to study migration in transgenic mouse models. (A) Expression of FoxP1, a marker for layer III/IV cortical neurons, was examined in pRb f/+; p107 +/-; Emx1-Cre + (WT), and pRb f/f; p107 -/-; Emx1-Cre + (DKO) brains at E17.5. Overlay includes Cux1 (blue - marker for layers II-IV) and Tbr1 (green - marker for layers V/VI). A population of cells expressing FoxP1 was observed in the intermediate zone of DKO embryos (arrow), but not WT embryos, with a concurrent decrease in the number of FoxP1+ neurons in layers II-IV of the cortex in DKO brains relative to WT brains (B). The white line indicates the delineation between the cortical plate and intermediate zone, which was determined using DAPI staining (not shown). (C) To trace the migration of a discrete population of late born neurons, WT and DKO embryos were electroporated with GFP at E14.5 and collected the brains at E18.5. Brains were co-stained with FoxP1 (red) and Tbr1 (blue) to show that GFP+ neurons are found almost exclusively in layer II in the WT brain but are delayed in the intermediate zone (IZ) and lower cortical layers in the DKO. (D) To determine if pRb and p107 regulate radial migration cell autonomously, pRb f/f; p107-/- and pRb f/+; p107-/+ embryos were electroporated with Cre-GFP at E13.5 and brains were collected at E17.5. The location of GFP+ neurons was quantified according to cortical layer. Layer II was defined as being above FoxP1 staining (red), layer III/IV was defined as within FoxP1 staining, above Tbr1 (blue) and layer V/VI was defined as within the Tbr1 positive layer (E). We show that a portion pRb and p107 deficient neurons fail to reach layer II but instead remain in layer III/IV. The figure has been modified from Svoboda et al., 2012. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51983/51983fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Embryo Age</td>
			<td>Voltage</td>
		</tr>
		<tr>
			<td>E12.5 &#8211; E13.5</td>
			<td>35V</td>
		</tr>
		<tr>
			<td>E14.5 &#8211; E15.5</td>
			<td>40V</td>
		</tr>
		<tr>
			<td>E16.5</td>
			<td>45V</td>
		</tr>
		<tr>
			<td>E17.5</td>
			<td>50V</td>
		</tr>
	</tbody>
</table>
Table 1: Electroporation voltage by embryo age.

Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

タンパク質の削除の誘導を介して<em&gt;子宮内で</em&gt;エレクトロポレーションは、トランスジェニックモデルにおいて神経細胞の移動の欠損を定義する

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

induction-protein-deletion-through-utero-electroporation-to-define

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

10.2K ビュー.  University of Ottawa. Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment. 

ビデオ: Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

子宮内エレクトロポレーションは、皮質ニューロンのサブセットで、遺伝子機能の研究のために初代神経培養した

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

神経科学

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

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 ...

Murine Neural Plate Targeting by In Utero Nano-Injection (NEPTUNE) at Embryonic Day 7.5

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously largely been restricted to the manipulation of embryonic stages post-neurulation. A protocol was developed to inject the amniotic cavity at embryonic day (E)7.5 and deliver lentivirus, encoding cDNA or shRNA, targeting &gt;95% of the neural plate and neural crest cells, contributing to the future brain, spinal cord, and peripheral nervous system. This protocol describes the steps necessary to achieve successful transduction, including grinding of the glass capillary needles, pregnancy verification, developmental staging using ultrasound imaging, and optimal injection volumes matched to embryonic stages. 
Following this protocol, it is possible to achieve transduction of &gt;95% of the developing brain with high-titer lentivirus and thus perform whole-brain genetic manipulation. In contrast, it is possible to achieve mosaic transduction using lower viral titers, allowing for genetic screening or lineage tracing. Injection at E7.5 also targets ectoderm and neural crest contributing to distinct compartments of the eye, tongue, and peripheral nervous system. This technique thus offers the possibility to manipulate gene expression in mouse neural-plate- and ectoderm-derived tissues from preneurulation stages, with the benefit of reducing the number of mice used in experiments.

Murine Neural Plate Targeting by In Utero Nano-Injection NEPTUNE at Embryonic Day 7.5

In this protocol, we describe how to inject the mouse amniotic cavity at E7.5 with lentivirus, leading to uniform transduction of the entire neural plate, with minimal detrimental effects on survival or embryonic development.

胎内ナノ注射(NEPTUNE)によるマウス神経板ターゲティング(胎内7.5日目)

Manipulating gene expression in the developing mouse brain in utero holds great potential for functional genetics studies. However, it has previously ...

発生生物学

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-、Hippocampus-、Thalamus-、Hypothalamus-、横中隔Nucleus-と線条体固有<em&gt;子宮内</em&gt; C57BL / 6マウスでのエレクトロポレーション

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

脳奇形の原因となる遺伝子を識別するアレイ CGH、全エキソーム配列と齧歯動物の子宮にエレクトロポレーションを組み合わせた新たな戦略

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング<em&gt;ウテロの</em&gt;エレクトロポレーション

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

子宮内エレクトロポレーション マウス大脳皮質における Cre lox 組換えを誘導します。

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブ イメージング用マウス萌芽期の脳の脊髄スライス培養

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

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子宮内エレクトロポレーションは、皮質ニューロンのサブセットで、遺伝子機能の研究のために初代神経培養した

使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

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

胎内ナノ注射(NEPTUNE)によるマウス神経板ターゲティング(胎内7.5日目)

Cortex-、Hippocampus-、Thalamus-、Hypothalamus-、横中隔Nucleus-と線条体固有

脳奇形の原因となる遺伝子を識別するアレイ CGH、全エキソーム配列と齧歯動物の子宮にエレクトロポレーションを組み合わせた新たな戦略

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング

子宮内エレクトロポレーションマウス大脳皮質における Cre lox 組換えを誘導します。

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブイメージング用マウス萌芽期の脳の脊髄スライス培養

タンパク質の削除の誘導を介して

要約

さらに動画を探す

この動画の章

子宮内エレクトロポレーションは、皮質ニューロンのサブセットで、遺伝子機能の研究のために初代神経培養した

使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

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

胎内ナノ注射(NEPTUNE)によるマウス神経板ターゲティング(胎内7.5日目)

Cortex-、Hippocampus-、Thalamus-、Hypothalamus-、横中隔Nucleus-と線条体固有

脳奇形の原因となる遺伝子を識別するアレイ CGH、全エキソーム配列と齧歯動物の子宮にエレクトロポレーションを組み合わせた新たな戦略

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

胚性マウス脳の器官型スライス培養における移行ニューロンの経時的共焦点イメージング

子宮内エレクトロポレーション マウス大脳皮質における Cre lox 組換えを誘導します。

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブ イメージング用マウス萌芽期の脳の脊髄スライス培養

子宮内エレクトロポレーションマウス大脳皮質における Cre lox 組換えを誘導します。

Ex 子宮エレクトロポレーションと gaba 作動性介在ニューロンの移行のライブイメージング用マウス萌芽期の脳の脊髄スライス培養