著者らは、Math1の、Ngn1ためにジェーン·ジョンソンに感謝したい、とPtf1a抗体およびpCAGためのコニーCepko :: GFPプラスミドと思います。この作品は、NIH R15 1R15HD059922-01によって資金を供給された。

1。エレクトロポレーションに先立って準備<ol><li>マキシプレップ（内閣総理 - それまたはキアゲン）でエレクトロポレーションのためのDNAを増幅する。 DNAの濃度は、効率的な取り込みは1 mg / mLの最小値である必要があります。 </li><li>マイクロ遠心チューブに1×PBS（リン酸緩衝生理食塩水）で0.01％ファストグリーンの5μlでDNAとミックスの495μLを削除します。 </li></ol> 2。胚の収穫<ol><li> CD-1マウス（Harlan）のタイムアウト交配を確立します。膣栓の有無をチェックして、膣栓が胚日（E）0.5として観察された日付を考えています。胚は11日プラグ（E11.5）を可視化した後に収穫されます。 </li><li> 70％エタノールで滅菌のツールを配置します。使用前に少なくとも1時間UV光で層流フードを扱う。スプレーフード、トレイを解剖し、70％エタノールで範囲を解剖。 37予熱1 X PBS℃で</li><li> E11.5に研究所によって承認された条件に従ってメスを安楽死させる動物のケアと使用（ICCUA）のional委員会。層流フードの解剖トレイを置きます。 70％エタノールで女性の腹部を下にスプレーします。 </li><li>腹腔を開き、解剖トレイに壁をピン。それが腹腔内にかかっているように子宮角を引き出します。 </li><li>慎重に子宮角の壁を切り開く。静かに胎盤から卵黄嚢の胚を削除するには、20ミリメートルのスプーン（ファイン科学ツール）を使用します。 2.2で予熱された滅菌1×PBS 10 mLであらかじめ入力さ100mmの組織培養皿に場所が胚。 </li><li>存在するすべての胚に対して、この手順を繰り返します。 </li></ol> 3。 E11.5胚のエレクトロポレーション（ 図1） <ol><li> 20 mmのスプーンを使用して、37に予熱された滅菌1XPBS 10mLの℃であらかじめ入力さ新しい100 mmディッシュに最初の胚を転送する</li><li>慎重に再0.05×0.02ミリメートルのチップ（ファイン科学ツール）で11 cmの鉗子を使用します。卵黄嚢に移動し、破棄します。 </li><li>位置胚ので、その背側は図1Aの漫画に似ているので、上を向いていること。静かに胚を保持するために7 mmの電極パドル（ハーバード装置）を使用します。電極は、後脳第四脳室レベルでの神経管の両側に配置する必要があります。心室は肉眼では見えて​​いますが、解剖顕微鏡を使用して正確なパドルの配置を容易にすることができます。パドルの位置決めは、エレクトロポレーション、DNAを受け取る領域を決定することが重要です。背側の胚後脳の下の菱形リップ（LRL）が必要な場合は、パドルは、彼らが直接に第四脳室の開口部の広い部分に隣接していることをそのような場所でなければなりません。 </li><li> 0.01パーセントの高速グリーンと混合したプラスミドDNAを描画する1ccのシリンジを使用しています。混合物の500μLには、少なくとも8月10日胚（3.5を参照）エレクトロポレーションのために十分でなければなりません。 </li><li>そっと穿刺roofplate OV1ccの注射器に接続されている25G 5月8日ツベルクリン針で第四脳室をerlyingと心室にDNA-染料混合物を注入します。成功した注射は、全体の心室システム（ 図1C）を充填DNA-染料の混合物によって特徴付けられる。一般的に注入されたDNA-色素混合物の量は50μL未満です。混合物の一部が胚を囲むPBS中に心室から漏れる傾向にあるように正確な金額は、胚の間で可変です。 DNA-色素混合物を提供する代替手段は、脳を覆っ​​て軟口蓋を穿刺による脳室系へのアクセスであるでしょう。再び、成功した注射は、全体の脳室系を充填DNA-染料の混合物によって特徴付けられる。 </li><li>電気パルスジェネレータ（BTX）および7 mmの電極パドルを使用して5つの方形波パルスを提供します。各パルスは、各パルスの間に500ミリ秒のパルス当たり50 V持続的な5 msです。正に帯電した電極に最も近い組織が鐸しますプラスミド上のe。 </li></ol> 4。胚の培養<ol><li>層流フードでは、10％ウシ胎児血清、5％ウマ血清、1％グルタミン、1％ペニシリン/予熱したストレプトマイシンを補充したDMEM/F12メディア2mLで12ウェル培養皿の外側の井戸を埋める37℃に培養条件は、デ·ディエゴや同僚から適応された15 </li><li>ピンセットで中央部（心臓の下）で層流フードのピンチ胚と胚の後方部分を削除します。 12ウェル培養皿の塗りつぶされた井戸の一つに前方部分に配置します。 </li><li>すべての胚に対して、この手順を繰り返します。文化の汚染を避けるために12ウェルプレートの外側の井戸を埋める。 </li><li> 5％CO 2、37℃インキュベーターで培養胚。エレクトロポレーションプラスミドの発現は24時間以内に観測する必要があります。 </li><li>より長い培養時間は、所望されるべき、2mLで新しい12ウェルプレートのうち、井戸を埋める4.1で使用されるメディア。滅菌スプーンで新しいプレートのウェルに胚を転送します。 37℃インキュベーターに戻してください。最大48時間の培養が可能です。 </li><li>希望の培養時間に達したときに、（下記参照）を分析のために胚を固定します。 </li></ol> 5。分析のための胚の準備<ol><li> 5分間、4℃で1×PBS中で胚を洗浄します。繰り返します。 </li><li> 4℃で2時間1XPBSで2％パラホルムアルデヒドの分析（PFA）のために胚を修正しました。 </li><li> 5分間、4℃で1×PBS中で胚を洗浄します。繰り返します。 </li><li> 4℃で一晩1 X PBS中30％スクロース中で胚を平衡℃に</li><li>ドライアイス/エタノール浴を用いて最適な切削温度（OCT）化合物の胚を埋め込むことができます。胚は-20℃で保存することができます</li><li> 30μm以下のセクションにクリオスタット（ライカ）と（VWR、Superfrostプラス）スライド上にマウントするのセクションの胚。 -20℃で保存</li></ol> 6。 IMMUnohistochemistry分析<ol><li> 16で説明したように免疫組織化学を行った。マウスα-MASH1（BD Biosciences）で1:100;ウサギα-Ngn1（ジェーン·ジョンソン）1:5000、ウサギα-Ptf1a（ジェーン·ジョンソンこの研究に用いた一次抗体希釈液は、ウサギα-GFP（Invitrogen社製）1:2500を含む）1:2500、ウサギ、Math1の（ジェーン·ジョンソン）1:100。 4℃で一晩染色トレイにスライドを平らな、試料面を上にしてインキュベートします。 </li><li>スライドは、複合顕微鏡（Olympus BX51）で分析した。 </li></ol> 7。代表的な結果 図1Aに模式的エレクトロポレーション実験を示しています。 図1Bは、操作の前にE11.5胚の矢状図を示します。プラスミドを含むCAGの注入後、同じ胚:: GFP 0.01％ファストグリーンは、 図1Cと一方的なGFPを示す代表的な不定胚に示されている背後脳24時間以下の培養での発現は、 図1Dに示されています。成功したエレクトロポレーションされてLRLの領域の範囲は可変であり、電極の位置に大きく依存するように見える。我々の研究では、エレクトロポレーション胚の65（80％）の52アウトに成功し、GFPを発現していることが判明した。それが（下記参照）の固定および免疫組織化学的分析は、次のいくつかのセクションに比べて局所領域におけるGFP陽性であった場合は、組織が正常にエレクトロポレーションされたものとみなされました。この基準を満たしていなかった胚は、エレクトロポレーションに失敗した試行と評価した。 エレクトロポレーション効率のさらなる評価は、第四脳室レベルでのエレクトロポレーションの胚の横断切片にGFPに対する免疫組織化学を行うことによって確かめることができる。 図2は、一方的な表示胚から代表的な連続切片GFP発現。 図2Aは、胚の左側には正極に向かったことを示しています理想的な回路図を示します。横断切片（ 図2Aの中央漫画で表さレベル）排他的に後脳組織（ 図2Cおよび2D）の左側にローカライズされたGFPの発現を明らかにした。 図2Cに示すように300μmの吻側以上のセクションの試験はGFPを発現する（ 図2B）をしないようLRLのエレクトロポレーション領域は、神経管の全体の前後軸を延長するものではありませんでした。 遺伝子発現の操作のためのこのアッセイの有用性は、内在性タンパク質の発現ドメインの安定性に依存しています。 LRLは、プロニューラル転写因子の差動式（1日）によって特徴づけられるユニークな前駆ドメインを有するとして特徴づけされています。これらの要因のサブセット（MASH1、Math1の、Ngn1と、Ptf1a）は、LRL、今後の研究16から18のテーマでprecerebellar神経サブタイプの仕様では彼らの提案、および/ ​​または特徴の役割のために分析のために選ばれました。すべての4つのタンパク質は、E11.5で16から18尾後脳に非常に特徴的な発現ドメインを持っています。我々は文化を置かれた胚のサイズが増加するのに失敗し、また脈絡叢上皮とLRLとE11.5とE12.5 5の間に発生するroofplate、形態学的イベントの陥入の生産を開始するために失敗したことを観察した。これらの観​​察結果に基づいて、これらの胚の正常な発達が停止または著しく遅延し、培養胚に匹敵するコントロールが、E11.5で教養のない胚でなければなりませんされていることを決定した。 文化やエレクトロポレーションは、内因性タンパク質のレベルを乱さないことを確認するために、我々はimmunohistochによって4つの異なるタンパク質を分析した私は、各マーカーの分析を行った胚の数を示しており、胚の割合は、通常のタンパク質レベルを保持していることを分析したエレクトロポレーションし、少なくとも24時間。 表の培養した34種類の胚でemistry（IHC）。 図3代表的なIHCデータ分析したタンパク質の2つ、MASH1（ 図3Aおよび3B）からとMath1の（ 図3Cおよび3D）。我々は、胚の大半はE11.5（ 図3Aと3C）での制御の胚に比べて（ 図3Bおよび3D）エレクトロポレーションと文化の後の式の正常なレベルを保持していることを観察した。重要なのは、これらのタンパク質の特性発現ドメインは乱されていませんでした。 <img src="/files/ftp_upload/3983/3983fig1.jpg" alt="図1"/> 図1。番目のE11.5における胚のエレクトロポレーション（A）の回路図eのエレクトロポレーション実験を行った。 E11.5の胚は、隔離され、0.01％ファストグリーンの発現プラスミドは、第四脳室に注入される。胚は、その後、電極パドルに挟まれたと前の培養液への注入前にE11.5胚の（B）矢のビューを配置されてから50 Vのパルスに供される。 （C）同じE11.5の胚は、0.01％ファストグリーンでのプラスミドの注入を後に。 （D）GFPの片側後脳の発現は、培養24時間後にE11.5胚で観察された。 MB-脳、4V-第四脳室。 <img src="/files/ftp_upload/3983/3983fig2.jpg" alt="図2"/> 図2。エレクトロポ組織におけるGFPの発現は、（A）左の漫画は、E11.5胚の周りに電極の配置を示しています。中央の漫画は、胚の左側をコードするプラスミドのGFPの取り込みおよび発現を示しています。右側の漫画は、Lで胚を通じて取得された理想的な横断面の模式図です。evelsは、中間漫画で黒い線で表される。文化の24時間後、エレクトロポレーションE11.5の胚を介して横断切片上でGFPの（B-D）免疫組織化学。矢印はroofplate（RP）がトラップを二次抗体を示しています。画像は10倍の倍率で撮影されています。示す各セクションの相対的なレベルは、（A）の真ん中の漫画を通る水平線で描かれています。 MB-脳、L 4V-第四脳室、RP-roofplate。 <img src="/files/ftp_upload/3983/3983fig3.jpg" alt="図3"/> 図3。低菱形リップの内 ​​因性タンパク質の発現は CAGとエレクトロポレーションした胚を培養ませんでしたE11.5胚（A、C）の横断面を比較しMASH1（AおよびB）またはMath1の（CおよびD）の免疫組織化学。 ：24時間（BとD）のGFPと培養した。画像は10倍の倍率で撮影した。 <table border="1"><tbody><tr><td> プロニューラルTranscriptiファクターで分析</td><td> 胚の数は、分析 </td><td> 通常の発現パターンを保持割合 </td></tr><tr><td> MATH1 </td><td> 15 </td><td> 86.7パーセント</td></tr><tr><td> MASH1 </td><td> 12 </td><td> 83.3パーセント</td></tr><tr><td> Ngn1 </td><td> 7 </td><td> 71.4パーセント</td></tr><tr><td> Ptf1a </td><td> 6 </td><td> 100パーセント</td></tr></tbody></table> LRLでノーマルプロニューラル転写因子ドメインを保持エレクトロポと培養胚の表Iの割合。

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hindbrain+rhombic+lip+is+comprised+of+discrete+progenitor+cell+populations+allocated+by+Pax6.">Hindbrain rhombic lip is comprised of discrete progenitor cell populations allocated by Pax6.</a> Neuron. 48, 933-947 (2005).</li><li>Hoshino, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ptf1a,+a+bHLH+transcriptional+gene,+defines+GABAergic+neuronal+fates+in+cerebellum.">Ptf1a, a bHLH transcriptional gene, defines GABAergic neuronal fates in cerebellum.</a> Neuron. 47, 201-213 (2005).</li><li>Yamada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+climbing+fiber+neurons+and+their+developmental+dependence+on.">Origin of climbing fiber neurons and their developmental dependence on.</a> Ptf1a. J. Neurosci. 27, 10924-10934 (2007).</li></ol>

利害の衝突が宣言されません。

本研究で提示したin vitroエレクトロポレーション技術は、効率的に妊娠12日未満の胚における遺伝子発現を操作するために利用することができる新たな方法論です。文化への胚の配置は、導入遺伝子の発現を可能にし、エレクトロポレーション胚をin vivoで維持する許可されているときに観察致死性を回避します。この手法は、エレクトロポレーションベースの研究のために以前?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>クライオスタット</td><td>ライカ</td><td> CM-1850 </td><td></td></tr><tr><td> Biologie先端Dumoxelはデュモン鉗子を治療</td><td>ファイン科学ツール</td><td> 11252から30 </td><td></td></tr><tr><td> 20ミリメートルMORIA穴あきスプーン</td><td>ファイン科学ツール</td><td> 10370から17 </td><td></td></tr><tr><td> ECM 830方形波エレクトロ·ジェネレータ</td><td> BTX（VWR） </td><td> 47745-928 </td><td></td></tr><tr><td>ハーバード装置7ミリメートルTweezertrodes *電極</td><td> BTX（フィッシャー） </td><td> BTX450165 </td><td></td></tr><tr><td>フィッシャーIsotemp CO 2インキュベータ</td><td>フィッシャー</td><td> 1325525 </td><td></td></tr><tr><td> NAPCO CO 2ガスレギュレータ</td><td>フィッシャー</td><td> 15497020 </td><td></td></tr><tr><td> 12ウェル組織培養プレート</td><td> BDファルコン（フィッシャー） </td><td> 877229 </td><td></td></tr><tr><td>ハイクローン液体培地DMEM/F-12（1:1）、L-グルタミンおよびHEPESを使って; 500mLに</td><td>サーモフィッシャーサイエンティフィック（フィッシャー） </td><td> SH3002301 </td><td></td></tr><tr><td>ハイクローン*ドナーウマ血清</td><td>サーモフィッシャーサイエンティフィック（フィッシャー） </td><td> SH3007402 </td><td></td></tr><tr><td>不活化ウシ胎児血清、修飾され、熱</td><td>インビトロジェン</td><td> 16140-063 </td><td></td></tr><tr><td> cellgro * 10,000 IUペニシリン、10,000μg/ mLのストレプトマイシン</td><td>メディアテック（フィッシャー） </td><td> MT-30-002-CI </td><td></td></tr><tr><td>ハイクローン* L-グルタミンL-グルタミン、200.85％NaClでの0mmの</td><td>サーモフィッシャーサイエンティフィック（フィッシャー） </td><td> SH3003401 </td><td></td></tr><tr><td>高速グリーン</td><td>フィッシャー</td><td> AC41053-0250 </td><td> 0.01パーセント</td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

菱形の唇は神経管と第四脳室（1日）のroofplate間の接合部における後脳に位置して胚の神経上皮である。菱形リップはrhombomere 1（R1）を包含し、小脳の神経細胞と神経細胞の多様な脳幹系統2-4を生じさせる下菱形リップ（LRL）を生成し、上部菱形リップ（URL）に細分することができます。 LRL誘導体は、聴覚蝸牛神経核の神経細胞のバランスとモータ制御5-8制御に関与しているprecerebellar核のものが含まれています。 LRLから神経新生は、胚日（E）9.5から16.5 5、9を含む大規模な一時的なウィンドウの上に発生します。別の神経系統は、この神経ウィンドウの間に明確な発達日間分裂細胞（または生まれている）のようなLRLから出てくる。 遺伝子発現コンストラクトのエレクトロポレーションに使用することができLRL前駆細胞における遺伝子発現を操作し、潜在的にこの地域の10月12日から生産ニューロンの運命を変更することができます。胎生12.5日以降10日12月14日に生まれた系統を操作するための非常に成功しました。 子宮内エレクトロポで前のE12.5に子宮内エレクトロポレーションで経由してマウスのLRL前駆細胞の遺伝子発現を変更すると、ために主に成功していない致死率は、第四脳室のroofplate、LRLにエレクトロされて​​いる外因性DNAを提供する上で必要なステップを穿刺に関連付けられています。しかし、多くのLRL派生した系統は、以前のE12.5 9よりLRLから生じる。これらの以前の生まれの系統は横網を構成するニューロンは、外部楔状束、小脳5に脊髄および大脳皮質からの入力を接続する機能precerebellarシステムの下オリーブ核が含まれています。 LRLの式を操作するためにE12.5未満の胚から、我々は、胚をエレクトロポレーション後に文化に置かれているのin vitroシステムを開発しました。 本研究では、E11.5でLRL前駆細胞の遺伝子発現を操作するための効率的かつ効果的な手法を提案する。広くアクティブなCAGプロモーターから駆動される緑色蛍光タンパク質（GFP）でエレクトロ胚は、再現性の文化の24時間後にGFPを発現した。このアッセイの重要な側面だけのため外来遺伝子の発現しないため、エレクトロポレーションおよび培養技術から、その結果二次的効果の変更されている遺伝子発現されています。それは、内在性遺伝子の発現パターンがエレクトロポレーションおよび培養胚で邪魔されずに残っていることが判明した。このアッセイは、過剰発現用プラスミドの導入によりE12.5より若い胚のLRLから出てきた細胞の運命を変更したり、の（RNAiを介して）をノックダウンするために利用することができる別のプロ神経転写因子。

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

本研究では開発について説明します。<em&gt; in vitroで</em妊娠中期の胚の下菱形リップにおける遺伝子発現の操作を可能にする&gt;エレクトロポレーション技術。

妊娠中期のマウス胚の低菱形リップの in vitroエレクトロポレーションで

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

Research

JoVE Journal

Neuroscience

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

10.3K ビュー.  University of Illinois at Springfield. 本研究では開発について説明します。 in vitroでエレクトロポレーション技術。

ビデオ: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

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

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

神経科学

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

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

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

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

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

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

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

開発マウス網膜の in vivoエレクトロポレーションで

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

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

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

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

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

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

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

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

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 ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

ニワトリ胚における軸索軌道とシナプスターゲットを追跡する後脳のエレクトロ

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

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 ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

不定冠詞<em&gt;インビトロ</em成体マウスの筋層間神経叢から分離腸ニューロンとグリアの&gt;準備

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

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

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

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

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