この作品は、独立行政法人理化学研究所脳科学総合研究センター（BSI）、ヒューマンフロンティアサイエンスプログラム（HFSP）（TSに授与）と理化学研究所ジュニアリサーチアソシエイトプログラム（JRA、MKに授与）独立行政法人理化学研究所脳科学総合研究センター（BSI）、ヒューマンフロンティアサイエンスプログラムによって資金を供給された（HFSP）（TSに授与）と理化学研究所ジュニアリサーチアソシエイトプログラム（JRA、MKに授与）は、この作業に資金を供給した。

1。注射のためのキャピラリーとDNA溶液の調製<ol><li>マキシプレップまたは同等の方法でプラスミドDNAを精製し、2〜3μg/μLの終濃度を行います。一定分量のDNAを100μg、速いグリーン染料（1％株式）とTE（10mMのトリスベース、1mMのEDTA溶液、pH8.0）を10μlの追加と1μgの/への注射のミックスの最終DNA濃度を確認するために100μlにボリュームを調整μL。プラスミドDNAの濃度を変更することができますが、個別にテストする必要がある、そのトランスフェクション効率、また、プラスミドの大きさに依存する、と。 </li><li>室温で14,000 rpmで5分間、DNA溶液をスピンし、残渣を除去する上清を集める。 </li><li>マイクロピペットプラーを使用してガラスキャピラリーtubeaのガラスキャピラリーチューブを引き出します。先端は20〜30μmの直径を持つように鉗子で先端を摘み取る。先端から800μmの1ミリメートルを測定し、外径がより小さい60μm以下（ 図1A）かどうかをチェック。 </li><li>マイクロマニピュレータにキャピラリーを接続し、鉱物油を詰めます。溶液中に先端水没、DNA溶液をキャピラリーを埋めて、DNA溶液を吸い上げる。 </li></ol> 2。白金電極を貼りここではそのような皮質（図1F）のような脳の表面的な地域、中にエレクトロに棒の白金電極（Nepe遺伝子、CUY611P2 - 1）を使用。 3。微小電極の調製脳の深い部分（すなわち、視床と視床下部）（図2B - E）にエレクトロに微小電極を使用してください。 <ol><li>一タングステンの終わり（負極用）とプラチナ（正極用）ワイヤが金メッキピンのコイルであり、パラフィルムで固定。綿棒の幹にワイヤーを挿入し、パラフィルム（図1B）で両端をラップ。 </li><li>それは800 20〜30μmの外径と60μmのミクロン- 1先端のmm（図1CおよびD）に達するまで均等に紙やすりでワイヤの先端をシャープに。 </li><li>絶縁用電線に磨き爪の薄い上着を適用します。マニキュアが乾燥した後、先端（先端から約200μm）からそれを削除するには、アセトンを浸した綿棒を使用してください。重要な注意：マウスの​​子宮への損傷を防ぐために、先端部800内に部分ミクロン- 1 mmは直径の70以下である（図1E）でなければなりません。 </li></ol> 4。手術の準備<ol><li>妊娠マウス（E9.5 - E15.5）を麻酔。私たちは、医薬品グレードのペントバルビタールナトリウムで麻酔を示しています（表II、グラム体重あたり50μgのは、腹腔内に注入し、5分間待つ）。麻酔薬としてペントバルビタールナトリウムの代替手段としては、ケタミン/キシラジン注射またはガス麻酔薬を（ガスは短時間の手順については、望ましいです）が含まれます。注射は、どの臓器を避けるために注意を払いながら行われるべき、それ以外の場合は子宮を損傷したり、動物を殺すことになります。成功するIPが​​完了すると、生存率は95％以上です。また、手術はオープンエアの環境で実行することができます。前に麻酔薬を確認するために彼らのつま先をつまんに動物による応答の欠乏のための試験、運転を開始した。 </li><li>かみそりの刃と50％エタノールを使用して腹部から毛を剃る。 betadineとアルコールのスクラブ（70％）を交互に肌を準備します。 </li><li>細かいはさみで腹部正中線で切開を加えると37の上に慎重にすべての子宮角を引き出す° C予め温めておいたリン酸緩衝生理食塩水（PBS）で湿らせた傷の周囲に配置されている綿のガーゼを、。 PBS時間のすべてを持つ子宮の潤いをキープ。 </li><li>人差し指と中指の間にフレキシブル光ファイバケーブルを保持し、子宮角の下に置きます。使用前に70％EtOHでクリーンケーブル。いいえ顕微鏡や拡大鏡は、視覚化は必要ありません。位置は、光ファイバの光ファイバケーブルと親指の間に子宮、そして近い子宮壁へ胚を押し上げる軽く絞る。 </li></ol> 5。 DNAとエレクトロポレーションの注入<ol><li>胚が置かれているときに、ガラスは、ターゲットの心室に慎重にキャピラリーとDNA溶液の約1μ（図2A）を注入挿入。 </li><li> electoporation用スティックの白金電極を使用してbrainIfの浅部へのエレクトロポレーションの場合は、子宮の外側に電極を配置し（表I）製造者の指示にしたがって提案された矩形波の電流パルスを適用する。脳の深部にusingForのエレクトロポレーション、nmicroのエレクトロポレーションした場合、eedle型電極が使用されます。 Iinsert DNAを注入心室と二つの電極の間に子宮や場所、対象領域への白金電極に微細なタングステン負極、その後提案された矩形波電流パルス（表I）（図2B）を適用します。 </li></ol> 6。ポストエレクトロポレーション<ol><li>そのORIGの子宮角のバックを置き、inalと場所、37℃で予め温めておいたPBS 500μLを加える。 </li><li> 9ミリメートルautoclipautoクリップ付き外科用縫合糸と密接な外層と内層を縫合。 </li><li> 2時間加熱パッドの上に置いて動物が麻酔からの回復を可能にする。このようなカルプロフェンまたはブプレノルフィンとして鎮痛薬は、術後の痛みのために投与することができます。 </li></ol> 7。代表的な結果： pCAG - EYFPの皮質エレクトロポレーションのいくつかの例は、異なる時点で棒の白金電極を使用して構築を図を図32に示す。脳は、E13.5、E14.5およびE15.5に電気穿孔、および出生後の日（P）6で収穫されています。 RORCのAThe ntibody染色GFP抗体染色により明らかにされており、両方のイメージが（ 図 3AおよびB）スーパー課されている層4とEYFPトランスフェクトされた細胞の位置の位置を明らかにする。標識された皮質の細胞は、エレクトロ皮質細胞の層は明らかに時間依存エレクトロポレーションは、皮質層固有のGOF / LOF可能に示唆している各々の実験（図32）、で異なっています。 細胞のトランスフェクションの胚と効率性の生存率は胚とエレクトロポレーションの場所の年齢によって異なります。サンプルは表1にリストされている、しかし、条件が舞台と脳の部分に応じて各エレクトロポレーション用に最適化する必要があります。 このようなスティックの白金電極を使用してある視床と視床下部などの深部組織へのエレクトロポレーションは、しばしば低効率（表1）を与える。この問題は、脳の深い部分に達すると、マイクロ電極のwhichelectrodesを、使用することで解決できます。図3図4に開発する間脳にpCAG - EYFPエレクトロポレーションの例。プラスミドDNA溶液は、E11.5で第3脳室に注入し、マイクロエレクトロ（ 図 3C4C）が続きます。エレクトロポレーション領域は、ほとんどの軸索のプロジェクトの皮質に（ 図 3B4B）視床に制限されています。皮質の層4への軸索投射はまた、全体のニューロンをfillesfills EYFP、可視化することができる。 （ 図 3A4A）。 <img src="/files/ftp_upload/3024/3024fig1.jpg" alt="図1"/> 図1キャピラリーの正しい形状を示す漫画の回路図。 （A）ガラスキャピラリーチューブは、一定の形状を作るためにマイクロピペットプラーを使用してプルアップされています。ヒントは、20〜30μmのように鉗子でピンチオフです​​。先端（赤ブラケット）から800μmの1ミリメートルを測定し、外径が60未満ミクロン（緑のブラケット）であることを確認してください。 （B）タングステンや白金線の一端​​は、金メッキ端子のコイルであり、パラフィルムで固定。その後20から30μmの外径と先端の1mmから60μmとなるようにその先端を作るためにサンドペーパーを使用してください。整形前のタングステンワイヤーの（C）画像。シェーピング後のタングステンワイヤーの（D）イメージ。マニキュアの薄い上着を適用した後のタングステンワイヤーの（E）イメージ。 Eのスケールバーは、CE用の10μm以下（最小スケール）です。 （F）スティックの白金電極（NEPAの遺伝子。CUY611P2 - 1）。 Fのスケールバーは2mmです。 <img src="/files/ftp_upload/3024/3024fig2.jpg" alt="図2"/> 外科的処置の図2。カートゥーンスキーマ。側脳室または大規模な胚（左側）にし、第3脳室や小さな胚（右側）への注入の（A）図。プラチナの棒電極（左側）からと針型電極（右側）によってelectrporationの（B）図。 <img src="/files/ftp_upload/3024/3024fig3.jpg" alt="図3"/> 図3。マウス終脳の開発は、E13.5（A）、E14.5（B）とE15.5（C）でpCAG - EYFPでエレクトロとP6で収穫された。脳は別々に冠状切片とGFPの抗体またはRORC抗体で染色し、画像がマージされます。 E13.5トランスフェ多くのレイヤ4のニューロンにおけるプラスミドpCAG - EYFPの（A）エレクトロポレーション。これは4のマーカーRORC層と重なる。レイヤ4のニューロンよりも表面的なE14.5トランスフェクション細胞を、少なくともプラスミドpCAG - EYFPの（B）エレクトロポレーション。 （C）E15.5エレクトロポレーションのラベルなどのレイヤ2 / 3など多くの表面的なニューロン。スケールバーは200μm。 <img src="/files/ftp_upload/3024/3024fig4.jpg" alt="図4"/> 図4。視床-皮質軸索（TCA）にEYFPのトランスフェクション。 （A）皮質層4のTCA末端は、GFP抗体染色により明らかにされます。 RORC抗体染色は層4の位置を明らかにするために使用されます。両方のイメージは、同じセクションから取得され、マージされます。 （B）視床へのエレクトロポレーションの位置はまた、視床11に制限されている表現、GFP抗体染色とRORC染色により示されています。 （C）視床のエレクトロポレーションおよびTCA投影のための漫画の回路図が示されています。スケールバーは200μm。

<ol>
	<li>Tabata, H., Nakajima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+utero+gene+transfer+system+to+the+developing+mouse+brain+using+electroporation:+Visualization+of+neuronal+migration+in+the+developing+cortex.">Efficient in utero gene transfer system to the developing mouse brain using electroporation: Visualization of neuronal migration in the developing cortex.</a> Neuroscience. 103, 865-872 (2001).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240, 237-246 (2001).</li><li>Fukuchi-Shimogori, T., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neocortex+patterning+by+the+secreted+signaling+molecule+FGF8.">Neocortex patterning by the secreted signaling molecule FGF8.</a> Science. 294, 1071-1074 (2001).</li><li>Kataoka, A., Shimogori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+controls+regional+identity+in+the+developing+thalamus.">Fgf8 controls regional identity in the developing thalamus.</a> Development. 135, 2873-2881 (2008).</li><li>Imayoshi, I., Shimogori, T., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hes+genes+and+neurogenin+regulate+non-neural+versus+neural+fate+specification+in+the+dorsal+telencephalic+midline.">Hes genes and neurogenin regulate non-neural versus neural fate specification in the dorsal telencephalic midline.</a> Development. 135, 2531-2541 (2008).</li><li>Shimogori, T., Banuchi, V., Ng, H. Y., Strauss, J. B., Grove, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signaling+centers+expressing+BMP,+WNT+and+FGF+proteins+interact+to+pattern+the+cerebral+cortex.">Embryonic signaling centers expressing BMP, WNT and FGF proteins interact to pattern the cerebral cortex.</a> Development. 131, 5639-5647 (2004).</li><li>Shimogori, T., Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+application+with+in+utero+electroporation+in+mouse+embryonic+brain.">Gene application with in utero electroporation in mouse embryonic brain.</a> Development Growth &#38; Differentiation. 50, 499-506 (2008).</li><li>Takahashi, Y., Watanabe, T., Nakagawa, S., Kawakami, K., Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+Embryology.">Avian Embryology.</a> Methods in Cell Biology. Vol 87, 271-271 (2008).</li><li>Feil, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-activated+site-specific+recombination+in+mice.">Ligand-activated site-specific recombination in mice.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 10887-10890 (1996).</li><li>Indra, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+mutagenesis+in+the+basal+layer+of+the+epidermis:+comparison+of+the+recombinase+activity+of+the+tamoxifen-inducible+Cre-ERT+and+Cre-ERT2+recombinases.">Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases.</a> Nucleic Acids Research. 27, 4324-4327 (1999).</li><li>Nakagawa, Y., O'Leary, D. D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+patterned+expression+of+orphan+nuclear+receptor+genes+ROR+alpha+and+ROR+beta+in+developing+mouse+forebrain.">Dynamic patterned expression of orphan nuclear receptor genes ROR alpha and ROR beta in developing mouse forebrain.</a> Developmental Neuroscience. 25, 234-244 (2003).</li></ol>

利害の衝突は宣言されません。

このプロトコルでは、我々は、異なる形の電極を使用して小さな胚の脳の限定された領域にpCAG - EYFPトランスフェクションの手法の利点を説明した。異なるプロモーターによるトランスフェクション効率の比較については、細胞型特異性1を示しており、以前に説明されています。しかし、制限が技術のためにある、トランスフェクションが唯一の一時的なもので、プラスミドに依存して変化するような、それは任意のセルの特異性を持っていないし、それがトランスフェクトされた細胞の数を制御することは困難です。 ThereforeHoweverは、他の手法でこのプロトコルを組み合わせると、さらに別の操作メソッドの可能性を提供します。最初に、プラスミドDNAに細胞特異的プロモーターを組み込むことはこのようなニューロン、グリア細胞やアストロサイトなどの細胞型に特異的なトランスフェクションを、できるようになります。第二に、トランスフェクションが一時的であるので、それは後の生活の中で遺伝子の機能を分析する科学者には適していません。そのため、プラスミドDNAがゲノムに組み込ませるトランスポゼースとの組み合わせは、安定なトランスフェクション8に変換されます。次に、テトラオンまたはtet -オフシステムの採用により、タイミングまたはセル8神経細胞の種類特異的な遺伝子発現を操作するようになります。別の方法として、タモキシフェン誘導性のCre - ER（T）リコンビナーゼ9およびレポーターマウス10でこのプロトコルを組み合わせることができます。 組織のこの広範なアクセシビリティが大幅に神経科学で使用されている実験的なデザインを変更します。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>裸のタングステン線径125μmの</td><td> AMシステム</td><td> 797600 </td></tr><tr><td>裸の白金線径125μmの</td><td> AMシステム</td><td> 767000 </td></tr><tr><td>スティック電極</td><td> NEPA遺伝子</td><td> CUY610P4 - 1 </td></tr><tr><td>ガラス毛細管</td><td> Stoelting </td><td> 50611 </td></tr><tr><td>マイクロマニピュレータ</td><td> KDサイエンティフィック</td><td> KDS310 </td></tr><tr><td>はさみ</td><td> ROBOZ </td><td> RS - 5865 </td></tr><tr><td>パルスジェネレータ</td><td> AMシステム</td><td> 2100 </td></tr><tr><td> 9mmのautoclip </td><td> ROBOZ </td><td> RS - 9260 </td></tr><tr><td>金メッキピン</td><td> WPI </td><td> 5482 </td></tr><tr><td> RORCの抗体</td><td>ペルセウスプロテオミクス</td><td> H3925 </td></tr></tbody></table>

mouse in utero electroporation: controlled spatiotemporal gene transfection

シグナル伝達分子と軸索ガイダンス分子、地元の遺伝子導入やノックアウト必要とされるなどの発展途上の脳の特定の領域で発現する遺伝子の機能を理解するために。ローカル領域にノックインまたはノックアウト遺伝子ターゲティングは、特定の骨の折れる、高価であるCREライン、、、時間がかかるとの組み合わせで実行することが可能です。したがって、単純なトランスフェクション法、 子宮内エレクトロポレーション法で 、短時間で実行できる、前のトランスジェニック動物の1,2の世代に候補遺伝子の可能な機能をテストするための便利なようになります。これに加えて、 子宮内エレクトロポレーションでは特定のCREのラインが存在しない脳の領域をターゲットとし、胚致死3,4が制限されます。ここで、我々は、脳の発達のターゲット領域に簡単で、便利な遺伝子導入用電極、2つの異なるタイプを組み合わせる子宮内エレクトロポレーションでの方法を提示する。最初に、光ファイバー、光ファイバケーブルを使用して、胚のユニークな保持方法は、対象となる脳の領域5,6に心室と針型電極の挿入にターゲットDNA溶液を注入するための小さな胚が（E9.5から）表示されるようになります。このような皮質領域のような脳のパターニングは、初期胚の段階で発生する、したがって、E9.5からこれらの初期のエレクトロポレーションは、全体面積のパターニングイベントを理解するために大きな貢献をする。第二に、キャピラリーの正確な形状は、毛細管を挿入することによって穴を作ることによって子宮の損傷を防ぎます。さらに、針電極の正確な形状は、タングステン及び白金線を使用して作成し、サンドペーパーを使用して先鋭化しマニキュア7、このプロトコルでは非常に詳細に記載されている方法で絶縁されています。このユニークな手法は、脳の制限区域へのプラスミドDNAのトランスフェクションを可能にし、小さな胚を電気穿孔することが可能になります。これは非常に初期の胚の段階で細胞分化、細胞移動、軸索誘導に取り組んでいる多くの科学者のための新しいウィンドウを開く、するのに役立ちます。また、この手法は、科学者は多くの地域固有のCREの行が関数のゲイン（GOF）または関数（LOF）の分析の損失のために存在していないような視床や視床下部などの脳の発達、の深い部分にプラスミドDNAをトランスフェクトすることができます。

Watch this Scientific Journal Video about Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection at JoVE.com

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

発展途上のマウス脳への遺伝子導入法は、ユニークな手術法と電極の特殊な形状を用いて記述されます。このユニークな手法は、脳の発達の研究に多くの神経科学者を支援する時間的および空間的にプラスミドDNAのトランスフェクションすることができます。

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 ...

mouse-utero-electroporation-controlled-spatiotemporal-gene

Research

JoVE Journal

Neuroscience

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

42.7K ビュー.  RIKEN Brain Science Institute. 発展途上のマウス脳への遺伝子導入法は、ユニークな手術法と電極の特殊な形状を用いて記述されます。このユニークな手法は、脳の発達の研究に多くの神経科学者を支援する時間的および空間的にプラスミドDNAのトランスフェクションすることができます。

ビデオ: Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

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 ...

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 ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

局所トランスジェニックラットの発生により<em&gt;子宮内</em&gt;エレクトロポレーションおよび<em&gt;インビボ</em&gt;生物発光スクリーニング

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

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 ...

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 ...

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 ...