Technical supported by Melanie Pfeifer and Nikolai Schmarowski (Institute for Microscopic Anatomy and Neurobiology, University Medical Center Mainz).

倫理声明：マウスの​​取り扱いおよび実験手順は、動物のケアのためのヨーロッパ、国および施設のガイドラインに従って行いました。 1. 子宮内エレクトロポレーション注意： 子宮内エレクトロポレーションでは 、以前に公開12,14のように行きました。したがって、この方法は、以下の（ 図1）に簡単に説明します。 <ol><li>準備<ol><li>以前に12を説明したように（1,5のpCAGGSを含む）ファストグリーン色のエンドトキシンを含まない高度なトランスフェクショングレードのプラスミド溶液を調製します。 </li><li>注射用（35°の角度）ホウケイ酸ガラスキャピラリー（0.8から0.9直径）を引いて、挽きます。 </li><li>楽器を滅菌します。 </li></ol></li><li>手術<ol><li>手術を開始する前に、鎮痛薬（カルプロフェン、4ミリグラム/ kg体重、皮下、24時間デポ）は、少なくとも30分を管理します。 </li><li>の長さにしてください35〜45分（ - Guedel 15に記載の外科手術用麻酔の段階-最大25〜30分ステージIII）の最大値に麻酔。 </li><li>イソフルラン（：2.8％、マスクを介した手術：2.5％チャンバー内の誘導）と時限妊娠マウスを麻酔。つま先ピンチに無反応によって麻酔を確認してください。 </li><li>麻酔中目の乾燥を防ぐために、眼軟膏を適用します。 </li><li> 7.5％ - イオジン溶液を用いて手術領域を滅菌し、露出しただけ手術領域を持つ（0.9％ベンジルアルコール食塩水で湿った）、滅菌ガーゼでマウスをカバーしています。 </li><li> （：1.5〜2センチメートル、筋肉の切開：1〜1.5センチの皮膚切開）メスで腹腔を開きカット。 </li><li>獣医スタッフや委員会の指示通りに温め、0.9％ベンジルアルコール食塩水または他の滅菌等張溶液で湿らせ乾燥から開か腹腔を保持します。 </li><li>リングピンセットを用いて（子宮角を慎重に抽出し、指で子宮に手を触れないでください秒）。 </li></ol></li><li> DNA及びエレクトロポレーションの注入。注：特定の領域を標的とするための正確な詳細は、セクション2に示されています。 <ol><li>準備されたホウケイ酸毛細血管を経由して、図1および2節で示したようにゆっくりと心室に着色されたDNA溶液を注入します。 </li><li>特殊な鉗子型白金電極を経由して対応する胚期（E14用例えば、36〜38 V）（インターバル周期長50ミリ秒、間隔休止950ミリ秒）のために適切な電圧を適用します。子宮壁の損傷を最小にするために、電圧の印加中にエレクトロポレーションパドルパルス。 </li></ol></li><li>ポストエレクトロポレーション/術後ケア<ol><li>慎重に腹腔内に子宮角を交換してください。 </li><li>別々に筋肉と皮膚切開を縫合。 </li><li>回復のための熱支持装置上でマウスの残りをしましょう​​。 </li><li>リカバリ中にマウスを監督（行動、フィード、水の消費量）、手術後4時間、24時間および48時間。必要に応じて、さらに2日間追加の鎮痛薬（カルプロフェン、4ミリグラム/ kg体重、24時間デポ）を適用。 </li></ol></li></ol>特定の脳領域の2トランスフェクション注：正極の位置は、DNA溶液（右または第三脳室）と正極の位置で満たされた心室を通る垂直中心線との間の角度として示されています。左心室を充填するときの位置がミラー反転されています。 図4に耳原始の位置が重要な基準点を提供する、示されています。 <ol><li>皮質領域のトランスフェクション（ 図2A、表1）。 <ol><li> 1.2節で説明したように手術を行います。皮質領域の子宮内エレクトロポレーションのためにE14の胚を使用しています。 </li><li>右側脳室に4μgのDNAを含有する1.5〜2μlのDNA溶液を注入準備されたホウケイ酸毛細血管を経由して。 <ol><li>穿刺部位上の目に見える血管が存在しないように慎重にリングピンセットを使用して胚を置きます。 </li><li>ゆっくりとラムダ縫合から約0.75ミリメートル冠状と矢状縫合（ 図3）から0.5ミリメートル、横DNA溶液を注入します。挿入深さが1.2ミリメートル（子宮壁から測定E14）であることを確認してください。シャープな境界と青緑色の着色のために確認してください。 </li></ol></li><li>特殊な鉗子型白金電極（間隔周期長50ミリ秒、間隔休止950ミリ秒）を介して電圧を適用します。 <ol><li> 3または5ミリの電極パドルを使用してください。 </li><li> 36-38 Vからまでの電圧を使用してください</li><li>電極耳原基のちょうど前方の中央に配置します。 </li><li> 、330から0°から脳梁膨大後部皮質の使用角度をトランスフェクトするために、運動野0-40°をトランスフェクトし、トランスフェクションするために、体性感覚皮質40〜95°をトランスフェクトします梨状皮質95-110°（ 図2A）。 </li></ol></li><li> 1.4節で説明したように、ポストエレクトロポレーション/術後ケアを行います。マウスを生け贄に捧げるとセクション3で説明したように4日間エレクトロポレーション（E18）後の胚を分析します。 </li></ol></li><li>海馬（ 図2B、 表1）のトランスフェクション。 <ol><li> 1.2節で説明したように手術を行います。海馬体の子宮内エレクトロポレーションのためにE15の胚を使用しています。 </li><li>準備されたホウケイ酸毛細血管を経由して右側脳室に4μgのDNAを含有する2μlのDNA溶液を注入します。 </li><li>特殊な鉗子型白金電極（間隔周期長50ミリ秒、間隔休止950ミリ秒）を介して電圧を適用します。 <ol><li> 3または5ミリの電極パドルを使用してください。 </li><li> 38-40の範囲の電圧を使用してください。 </li><li>負極耳原基のちょうど前方の中心と中心Oを配置耳の原基の途中で正極F​​。 </li><li>歯状回はCA1 250〜275°（ 図2B）をトランスフェクトするために、CA2 230-250°をトランスフェクトするために、アンモン角（CA）3 210〜230°をトランスフェクトするために、190から210°の角度を使用してトランスフェクトします。 </li></ol></li><li> 1.4節で説明したように、ポストエレクトロポレーション/術後ケアを行います。第3章で説明したように（出生後、P21）は、完全な海馬形成後に分析を実行します。 </li></ol></li><li>横中隔核および線条体のトランスフェクション（ 図2C、 表1） 注意：角度は海馬をトランスフェクトするためのものと重複します。効果的に横中隔核と不要な海馬のトランスフェクション以前の胚の段階（E12）のない線条体をトランスフェクトするために使用されなければなりません。 <ol><li> 1.2節で説明したように手術を行います。横中隔核とstriatの子宮内エレクトロポレーションのためにUM E12の胚を使用しています。 </li><li>準備されたホウケイ酸毛細血管を経由して右側脳室に4μgのDNAを含む1μlのDNA溶液を注入します。 </li><li>特殊な鉗子型白金電極（間隔周期長50ミリ秒、間隔休止950ミリ秒）を介して電圧を適用します。 <ol><li> 0.5ミリメートルの電極を使用してください。 </li><li> 33 Vを使用します</li><li>耳の原基のレベルで電極の中心を配置します。 </li><li>線条体170〜240°（ 図2C）をトランスフェクトするために、100〜170°の横中隔核使用角度をトランスフェクトします。 </li></ol></li><li> 1.4節で説明したように、ポストエレクトロポレーション/術後ケアを行います。マウスを生け贄に捧げるとセクション3で説明したように6日、エレクトロポレーション（E18）後の胚を分析します。 </li></ol></li><li>視床と視床下部のトランスフェクション（ 図2D、 表1） <ol><li> 1.2節で説明したように手術を行います。 Uでの場合視床と視床下部利用E12-E13胚のTEROエレクトロ。 </li><li>準備されたホウケイ酸毛細血管を経由して（左または右）側脳室に4μgのDNAを含有する1〜1.5μlのDNA溶液を注入します。解決策は、第三脳室中に拡散されるまで、2〜3分間待ちます。あるいは、直接第三脳室に注入。 注意：これは、特異性を向上させますが減少し、生存率（損傷の危険性が高い）を引き起こす可能性があります。 </li><li>特殊な鉗子型白金電極（間隔周期長50ミリ秒、間隔休止950ミリ秒）を介して電圧を適用します。 <ol><li> 0.5または3mmの電極を使用してください。 </li><li> 33-35 Vからまでの電圧を使用してください</li><li>ちょうど耳の原基を後方電極の中心を配置します。 </li><li>視床下部90-180°（ 図2D）をトランスフェクトするために、25〜40°から視床の使用角度をトランスフェクトします。 </li></ol></li><li> DESなどのポストエレクトロポレーション/術後のケアを行います1.4節でcribed。マウスを生け贄に捧げるとセクション3で説明したように6日、エレクトロポレーション（E18）後の胚を分析します。 </li></ol></li></ol> 3.灌流固定<ol><li>準備<ol><li> 37°Cまで暖かい4％パラホルムアルデヒド（PFA）。 </li><li> PBSで50mlの注射器（4℃）と温めPFAと第2のシリンジを埋めます。泡を避けてください。 </li><li>三方活栓にシリンジを接続します。翼の輸液セット（蝶）の三番目の端を接続します。 </li><li> PBSで翼の輸液セットをフラッシュします。 </li></ol></li><li>灌流<ol><li>深く皮下塩酸ケタミン（60mg / kg）を適用およびキシラジン（7.5 mgの/ kg）でマウス（妊娠中や出産後のトランスフェクト）を麻酔。 </li><li>つま先ピンチに無応答性により、外科的寛容の段階を確認してください。 </li><li>ちょうど胸郭の下に腹腔を開きます。 </li><li>慎重に横隔膜をカット。 </li><li>慎重電子のカットを経由して胸郭を開きます鎖骨までACHサイト。 </li><li> （心尖から開始）左心室に蝶の先端を挿入します。 </li><li>左心耳をカットします。 </li><li>ゆっくりPBS（約10ml）でマウスを灌流。 </li><li>三方活栓を切り替えます。 </li><li> 5〜10ミリリットルの4％PFAでマウスを灌流。 </li><li>はさみを使用して、マウスを刎ねると大後頭孔から始まる脳を解剖。以前に発表された16のように解剖を行います。 </li><li> 4％PFA中で2日間の脳をPostfixate。暗所で4℃で脳をしてください。 </li></ol></li></ol> 4.免疫組織化学<ol><li> （ビブラトームで例えば ）70μmのセクションを生成します。 </li><li>オプション：抗体染色と蛍光を強化します。 <ol><li> RTで1時間切片をブロック（0.1から0.2％のTriton X-100、5％正常ヤギ血清） </li><li>適切な抗体（：1,000抗GFP、1）でO / Nインキュベート</li><li>ウォッシュ30.1Mリン酸緩衝液で-5倍。 </li><li>適切な蛍光結合二次抗体（：1,000アレクサフルオロ488標識抗ウサギIgGを-、1）で3-5時間インキュベートします。 </li><li>オプション：1分（0.1 Mリン酸緩衝液中でDAPI、0.2グラム/ミリリットル）4-6 - diaminodino -2-フェニルインドールで対比染色。 </li><li>適切な蛍光顕微鏡で蛍光を分析します。 </li></ol></li></ol>

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The authors have nothing to disclose.

This protocol describes in detail how to transfect 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 of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

3独立したグループ1-3により2001年に最初の記述以来I N 子宮内エレクトロポレーションは、齧歯動物の中枢神経系における遺伝子発現を分析するために広く使用されている標準的なツールとなっています。継続的に技術、まだ時間とお金がかかり、そのシンプルさに起因する子宮内エレクトロポレーションのアピールを改善するにもかかわらず、であるノックアウトマウスの作製に比べ。だから、 子宮内エレクトロポレーションは、高速かつ効率的gain-や機能喪失研究4を可能にします 。 脳の領域をトランスフェクトするために、負に帯電したプラスミドを含有する溶液を脳室に注入されます。電気パルスの間に、負に荷電したDNAは、正極に向かって移動するので、トランスフェクトされた領域は、正極の位置を変更することによって簡単に選択することができます。これは、頻繁に、中枢神経系の多数の領域がTAであることが示されています3,5-8を rgeted。例えば、最近の研究では、海馬、梨状皮質や線条体9-11の具体的なトランスフェクションを示します。しかし、適切な位置についての情報は、多くの場合のみ、ほとんど標準化されず、常に異なるマウス系統に転送することは容易ではありません。 特定胚の段階のトランスフェクションは、些細なほど遠いです。 子宮内エレクトロポレーションの特定のためのセットアップを選択する際に多くの影響を与える要因を考慮する必要があります。まず、最適にそれぞれの胚の段階をトランスフェクトするために、適切な電圧についての知識が必要とされています。低電圧は、トランスフェクション効率2,3,12を減らす一方、高電圧は、生存率を低下させます。また、電極パドルのサイズが減少した特異性には大きすぎる結果であるか、心臓の鼓動4,12,13の影響に起因する死亡を引き起こす可能性があり、電極パドルを使用するため、重要な役割を果たしています。適用されました電圧と大きさと電極パドルの位置が考慮すべき最も重要な機能であるが、DNA-液の塗布量のように、エレクトロポレーションの結果に影響を与えるさらなる要因もあります。 我々は、C57BL / 6 マウス 12の様々な脳の領域の迅速かつ効率的なトランスフェクションを可能にする詳細なプロトコルを開発しました。このプロトコルでの電圧についての詳細な情報を使用する、強化特異用電極パドルのサイズが提供されます。さらに、心室についての情報は、プラスミド溶液の量についての勧告に沿って満たされると、電極の位置が供給されています。マップ内の詳細な位置情報の表示、これらの位置のさらなる可視化は、脳梁膨大後部皮質の子宮内エレクトロポレーション、運動皮質、体性感覚皮質、梨状皮質、トンで簡単な特定かつ効率的に可能彼は1-3、歯状回、線条体、横中隔核、視床および視床下部ammonisコルニュ。

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			of the University Medical Center Mainz</td>
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			<td height="21" style="height:21px;">gauze (ES-Kompressen)</td>
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			<td>Ethicon Johnson &#38; Johnson</td>
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			<td>Fine Science Tools</td>
			<td>11103-09</td>
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			<td height="21" style="height:21px;">Adson Forceps-Serrated Straight 12 cm</td>
			<td>Fine Science Tools</td>
			<td>1106-12</td>
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			<td height="21" style="height:21px;">IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td>
			<td>Fine Science Tools</td>
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			<td>Fine Science Tools</td>
			<td>14012-15</td>
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			<td height="21" style="height:21px;">Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
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			<td height="21" style="height:21px;">Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td>
			<td>Fine Science Tools</td>
			<td>12565-14</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Elektroporator CUY21 SC&#160;</td>
			<td>Nepa Gene Co.</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">FST 250 Hot Bead Sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td></td>
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			<td height="21" style="height:21px;">Microgrinder EG-44</td>
			<td>Narishige</td>
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			<td height="21" style="height:21px;">P-97 Micropette Puller</td>
			<td>Sutter Instrument Company</td>
			<td>P-97</td>
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			<td height="21" style="height:21px;">Platinum electrodes 650P 0.5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P0.5</td>
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			<td height="21" style="height:21px;">Platinum electrodes 650P 3 mm</td>
			<td>Nepagene</td>
			<td>CUY650P3</td>
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			<td height="21" style="height:21px;">Platinum electrodes 650P 5 mm</td>
			<td>Nepagene</td>
			<td>CUY650P5</td>
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			<td height="21" style="height:21px;">Platinum electrodes 650P 10 mm</td>
			<td>Nepagene</td>
			<td>CUY650P10</td>
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			<td height="21" style="height:21px;">Anesthesia system</td>
			<td>Rothacher-Medical GmbH</td>
			<td>CV-30511-3 Vapor 19.3</td>
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			<td>HP-1M</td>
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			<td>Rothacher-Medical GmbH</td>
			<td>TCAT-2LV</td>
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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.

図2は 、脳梁膨大後部皮質に開発途上地域の子宮内エレクトロポレーション内の特定の例を示し、運動野、体性感覚皮質、梨状皮質、コルニュは1-3 ammonis、歯状回、線条体、側方中隔核、視床および視床下部。トランスフェクションの結果は、次の推奨される角度（ 図2）に示されています。 生体内での角度のより良い視覚化のための電極（0.5ミ?...

Watch this Scientific Journal Video about Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse at JoVE.com

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 ...

cortex-hippocampus-thalamus-hypothalamus-lateral-septal-nucleus

Research

JoVE Journal

Neuroscience

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

27.8K ビュー.  University Medical Center, JGU Mainz. 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.

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

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

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 ...

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 ...

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, ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

機能と神経病理学的評価の c57bl/6 マウスの実験的自己免疫性視神経炎を誘導するための単純なアプローチ

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

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 ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

人工内耳手術と c57bl/6 マウスに電気的誘発脳幹の応答録音

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

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

要約

さらに動画を探す

この動画の章

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

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

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

局所トランスジェニックラットの発生により

マウスの遺伝子操作は、視床下部を介して開発

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

機能と神経病理学的評価の c57bl/6 マウスの実験的自己免疫性視神経炎を誘導するための単純なアプローチ

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。

人工内耳手術と c57bl/6 マウスに電気的誘発脳幹の応答録音

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

要約

さらに動画を探す

この動画の章

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

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

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

局所トランスジェニックラットの発生により

マウスの遺伝子操作は、視床下部を介して開発

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

機能と神経病理学的評価の c57bl/6 マウスの実験的自己免疫性視神経炎を誘導するための単純なアプローチ

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。

人工内耳手術と c57bl/6 マウスに電気的誘発脳幹の応答録音

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。