Name: cortex-, hippocampus-, thalamus-, hypothalamus-, lateral septal nucleus- and striatum-specific in utero electroporation in the c57bl/6 mouse
Uploaded: 2016-01-18T14:00:00.000+00:00
Duration: 6 min
Description: 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

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 regions and therefore the detailed information needed for specific transfections is only scarcely standardized. 
The position and the size of the electrode as well as the applied voltage for the corresponding embryonic stage, which are the critical steps for effective and specific transfections, are precisely described. The provided angle-map helps to orientate the electrodes for specific electroporation. The locations of the electrodes are additionally shown on an embryo in the uterus, an embryo in the amniotic sac and also on a pure, dissected embryo. All of this is intended to help in identifying the correct positions of the electrodes. 
Usage of the appropriate voltage is a central aspect in the provided protocol. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency 2,3,12. Voltage recommendations given in this protocol are specific for C57BL/6 mice and slightly differ from these for CD12,3,12. CD1 mice tolerate higher voltages than C57BL/6 mice and therefore higher voltages might be used for transfecting CD1 mice12. No data exist for other mouse strains, but the standards might need some modifications. 
A further central point is the size of the electrode. The usage of a smaller electrode, rather than a bigger one, is always recommended for experiments where specificity is important. Smaller electrodes enhance specificity4,12. If the specificity of the transfection is not the main point, especially for inexperienced experimenters, bigger electrodes enhance the chance of hitting the desired region. Thereby one has to keep in mind that electrodes covering too much of the embryo, might affect the heart rhythm and lead to reduced survival. For example a 10 mm electrode covers the whole E12 embryo and therefore is not recommended for transfecting E12 embryos, even if specificity is not the primary goal. 
The stages used in this protocol were chosen exemplary. Considering the fact that progenitors that are transfected on a certain time point give rise to a particular type of neuron the embryonic stages for transfection can be adapted to the specific aim of the study. For instance during cortical development, neurons are born at different time points, forming the six cortical layers in a sequential manner. Hence, depending of the time point of transfection the transfected progenitors give rise to neurons of a specific layer2,17.
For a successful transfection the following points also should be observed. First, the careful preparation and injection of the glass capillaries play a crucial role for the survival of the embryos and therefore for effective transfection. Second, the selection of an appropriate DNA concentration can also be very important and might have to be specifically adapted for different vectors18. Third, application of high voltages affects neighboring embryos, and therefore transfection of neighboring embryos might reduce the survival of these embryos. Further it has also been recommended to avoid the embryos closest to the vaginal duct, because the damage of these embryos might lead to abortion18.
Besides great advantages the technique of in utero electroporation has its drawbacks, which should be taken into consideration while designing experiment. First the regions, which can be efficiently transfected, are manly restricted to areas surrounding the ventricles. This is due to the fact that the plasmid-solution has to be injected into a ventricle. Further, even with all the details provided in this protocol it is not possible to transfect all cells out of a specific region. Even the number of transfected cells is hardly controllable. The analysis of the gene function is not restricted to embryonic stages. Adult mice can also be analyzed because the transfection of postmitotic neurons lasts at least up to 4 months13, but is not passed to the next generation. 
Further specifications, such as specifically electroporating thalamic and hypothalamic nuclei or temporally addressing cortical layers can additionally broaden the scope. The combination of genetically engineered mice, or inducible systems in with the in utero electroporation also additionally enhances possible applications. 
With all the given facts, this protocol facilitates specific transfections and therefore reduces the number of animal used. Standardization of the protocol additionally enhances the comparability of the obtained results.

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。

<table><tbody><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Fast Green</td><td>Roth</td><td>0301.1</td><td></td></tr><tr><td>pCAGGS</td><td>Addgene</td><td></td><td></td></tr><tr><td>borosilicate glass capillaries (0.8-0.9 mm diameter)</td><td>Wold Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Isoflurane (Forene)</td><td>Abbott</td><td>PZN 4831850</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>approval number: 400684.00.00</td><td></td></tr><tr><td>eye ointment (Bepanthen Augen und Nasensalbe)</td><td>Bayer&amp;nbsp;</td><td>PZN 01578681</td><td></td></tr><tr><td>0.9% benzyl alcohol 0.9% saline solution</td><td>Pharmacy&lt;br/&gt; of the University Medical Center Mainz</td><td></td><td></td></tr><tr><td>gauze (ES-Kompressen)</td><td>Hartmann</td><td>407835</td><td></td></tr><tr><td>sterile 5-0 Perma-Hand Silk Suture</td><td>Ethicon Johnson &amp;amp; Johnson</td><td>K890H</td><td></td></tr><tr><td>ring forceps 1/ 1.5 mm</td><td>Fine Science Tools</td><td>11101-09</td><td></td></tr><tr><td>ring forceps 4.8/ 6 mm</td><td>Fine Science Tools</td><td>11106-09</td><td></td></tr><tr><td>ring forceps 2.2/ 3 mm</td><td>Fine Science Tools</td><td>11103-09</td><td></td></tr><tr><td>Adson Forceps-Serrated Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>IrisScissors-Delicate Straight-Sharp/Blunt 10 cm</td><td>Fine Science Tools</td><td>14028-10</td><td></td></tr><tr><td>Mayo-Stille Scissors-Straight 15 cm</td><td>Fine Science Tools</td><td>14012-15</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm</td><td>Fine Science Tools</td><td>12565-14</td><td></td></tr><tr><td>Elektroporator CUY21 SC&amp;nbsp;</td><td>Nepa Gene Co.</td><td></td><td></td></tr><tr><td>FST 250 Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Microgrinder EG-44</td><td>Narishige</td><td></td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Platinum electrodes 650P 0.5 mm</td><td>Nepagene</td><td>CUY650P0.5</td><td></td></tr><tr><td>Platinum electrodes 650P 3 mm</td><td>Nepagene</td><td>CUY650P3</td><td></td></tr><tr><td>Platinum electrodes 650P 5 mm</td><td>Nepagene</td><td>CUY650P5</td><td></td></tr><tr><td>Platinum electrodes 650P 10 mm</td><td>Nepagene</td><td>CUY650P10</td><td></td></tr><tr><td>Anesthesia system</td><td>Rothacher-Medical GmbH</td><td>CV-30511-3 Vapor 19.3</td><td></td></tr><tr><td>Heating plate</td><td>Rothacher-Medical GmbH</td><td>HP-1M</td><td></td></tr><tr><td>Temperature Controller 220V AC</td><td>Rothacher-Medical GmbH</td><td>TCAT-2LV</td><td></td></tr></tbody></table>

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ミリメートル）の位置も（羊膜嚢で図5、 子宮内胚、胚、解剖胚）の胚に表示されます。概要表1を提供するために特定のトランスフェクションのための関連する事実をまとめました。 また、電極のサイズは特異性（ 図6）のために重要な役割を果たしていることが実証されました。電極パドルの大きなサイズは、ラにつながります典型的な図 6に示すようにrgerは、領域をトランスフェクトした。さらに、胚のあまり大きすぎるカバーされている電極を、それによって胚の心臓の鼓動（ 図7）に影響を与える危険性を高めます。例えば10ミリメートルの電極パドル全体E12胚（ 図7）を覆っています 。 <img alt="図1" src="/files/ftp_upload/53303/53303fig1.jpg" /> 子宮内 エレクトロポレーション では 、図1。 子宮内エレクトロポレーションのパフォーマンスの基本的な手順が示されています。 （A）は、既にイソフルランで麻酔したホットプレート上に配置されたマウスで、手術の全体の設定を表示します。 （B）手術領域の消毒には、マウスの腹腔（C）を開く前に、重要なステップです。 （D）DNAを含む溶液を、高速Gで着色されています成功した注射の可視化を可能にREEN、（E、F）。側脳室への成功注入は三日月形スポットになります。 （G）は、注射後に適切な電圧をピンセット型の白金電極を介して印加されます。 （H）を慎重に手術創を縫合し腹腔内に子宮を交換した後。 <a href="https://www.jove.com/files/ftp_upload/53303/53303fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53303/53303fig2.jpg" /> マウス大脳皮質、海馬、横中隔核、線条体、視床と視床下部の特定の領域のエレクトロポレーションのための図2アングルマップ。正と負極のための適切な位置は、概略図に示されています。 （ACについて</stro<html> NG&gt;）の位置は、右心室へのDNA溶液を注入するために示されています。左心室に注射するため、角度は、ミラー反転されています。特定のトランスフェクションは、組織学的に検証しました。 （A）は、皮質領域（脳梁膨大後部皮質（RSC）、運動皮質（MC）、体性感覚皮質（SSC）と梨状皮質（PC）をトランスフェクトするための正と負極の位置を表示します。（B）が表示します海馬形成をトランスフェクトするために正と負極の位置（コルニュは1-3 ammonis（CA1、CA2、CA3）および歯状回（DG）。（C）は、線条体をトランスフェクトするために正と負極の位置を示しています（STR）と横中隔核（LS）。（D）は、視床（TH）および視床下部（HY）をトランスフェクトするための電極の適切な位置を示している。Baumgart J.＆カイツブリN.、2015年12から適応。<html> PS：//www.jove.com/files/ftp_upload/53303/53303fig2large.jpg「ターゲット= &quot;_空白&quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図3" src="/files/ftp_upload/53303/53303fig3.jpg" /> 図3穿刺部位は、DNA溶液の注入のための最適な位置であります 約ラムダ縫合および0.5ミリメートルから冠状0.75ミリメートルは、矢状縫合から側方。 <a href="https://www.jove.com/files/ftp_upload/53303/53303fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53303/53303fig4.jpg" /> 耳の原基の図4の位置。耳原基の位置が明らかに（矢印）が定義されています。NK &quot;&gt;この図の拡大版をご覧になるにはこちらをクリックしてください。 <img alt="図5A" src="/files/ftp_upload/53303/53303fig5A.jpg" /><img alt="図5B" src="/files/ftp_upload/53303/53303fig5B.jpg" /><img alt="図5C" src="/files/ftp_upload/53303/53303fig5C.jpg" /><img alt="図5D" src="/files/ftp_upload/53303/53303fig5D.jpg" /> 胚の角度の図5.可視化。より良い視覚化のための皮質領域の特定のトランスフェクション（脳梁膨大後部皮質、運動野、体性感覚皮質、梨状用正（青のプラス記号で示す）と負極のための適切な位置皮質）、海馬領域（アンモン角（CA）1-3、歯状回（DG））、羊膜嚢及び解剖胚（すべてE15,5）の ​​横中隔核、線条体、視床と視床下部は、 子宮内の胚に表示され、胚。 <a href="https://www.jove.com/files/ftp_upload/53303/53303fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図6" src="/files/ftp_upload/53303/53303fig6.jpg" /> 電極のサイズに応じて、トランスフェクションの図6特異。電極パドルのサイズの増加は、トランスフェクションの特異性を低下させます。これは、後期胚形成期（E17、下）で特に若い胚の段階（E12、トップ）で明らかなだけでなく、表示されています。 Baumgart J.＆カイツブリN.、2015年12から適応。 <a href="https://www.jove.com/files/ftp_upload/53303/53303fig6large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a><img alt="図7" src="/files/ftp_upload/53303/53303fig7.jpg" /> E12胚の大きさに比べて、電極パドルの図7.サイズ。胚のあまり大きすぎるカバーであり、したがって、心臓のリズムの影響のリスクを増加させる電極。例えば、（右側）10ミリメートルの電極は、特異性が主な目的でない場合であっても、全体のE12胚をカバーするため、お勧めできません。 Baumgart J.＆カイツブリN.、2015年12から適応。 <a href="https://www.jove.com/files/ftp_upload/53303/53303fig7large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 標的領域 </td><td> 角度 </td><td colspan="2"> 電極の位置 </td><td> 胚の年齢 </td><td> DNAのamouNT /濃度</td><td> 電圧 </td><td> 電極の大きさ </td></tr><tr><td>脳梁膨大後部皮質</td><td> 330° - 0° </td><td colspan="2">耳の原基の電極の中心はちょうど前方</td><td> E14（E11-E17） </td><td> 1.5 2μL/ 2.7- 2μgの/μL </td><td> 36- 38 </td><td> 3 5ミリメートル</td></tr><tr><td>モーターのCortex </td><td> 0° - 40° </td><td colspan="2">耳の原基の電極の中心はちょうど前方</td><td> E14（E11-E17） </td><td> 1.5 2μL/ 2.7- 2μgの/μL </td><td> 36- 38 </td><td> 3 5ミリメートル</td></tr><tr><td>体性感覚皮質</td><td> 40° - 95° </td><td colspan="2">耳の原基の電極の中心はちょうど前方</td><td> E14（E11-E17） </td><td> 1.5 2μL/ 2.7- 2μgの/μL </td><td> 36- 38 </td><td> 3 5ミリメートル</td></tr><tr><td> Piriform個のCortex </td><td> 95° - 110° </td><td colspan="2">耳の原基の電極の中心はちょうど前方</td><td> E14（E11-E17） </td><td> 1.5 2μL/ 2.7- 2μgの/μL </td><td> 36-3 8 </td><td> 3 5ミリメートル</td></tr><tr><td>アンモン角 （CA）1 </td><td> 250° - 275° </td><td colspan="2">耳の原基の途中で正極のセンター</td><td> （E14-）E15 </td><td> 2μL/ /μlの2μgの</td><td> 38- 40 </td><td> 3 5ミリメートル</td></tr><tr><td> CA2 </td><td> 230° - 250° </td><td colspan="2">耳の原基の途中で正極のセンター</td><td> （E14-）E15 </td><td> 2μL/ /μlの2μgの</td><td> 38- 40 </td><td> 3 5ミリメートル</td></tr><tr><td> CA3 </td><td> 210° - 230° </td><td colspan="2">耳の原基の途中で正極のセンター</td><td> （E14-）E15 </td><td> 2μL/ /μlの2μgの</td><td> 38- 40 </td><td> 3 5ミリメートル</td></tr><tr><td>歯状回</td><td> 190° - 210° </td><td colspan="2">耳の原基の途中で正極のセンター</td><td> （E14-）E15 </td><td> 2μL/ /μlの2μgの</td><td> 38- 40 </td><td> 3 5ミリメートル</td></tr><tr><td>線条体</td><td> 170° - 240° </td><td colspan="2">耳の原基のレベルでの電極のセンター</td><td> E12 </td><td> 1μL/ 4μgの/μL </td><td> 33 </td><td> 0.5ミリメートル</td></tr><tr><td>横中隔核</td><td> 100° - 170° </td><td colspan="2">耳の原基のレベルでの電極のセンター</td><td> E12 </td><td> 1μL/ 4μgの/μL </td><td> 33 </td><td> 0.5ミリメートル</td></tr><tr><td>視床</td><td> 25° - 40° </td><td colspan="2"> electodesのセンターは、ちょうど耳のPRIを後方mordia </td><td> E12- E13 </td><td> 1〜1.5μL/ 4から2.7μgの/μL </td><td> 33- 35 </td><td> 0.5〜3ミリメートル</td></tr><tr><td>視床下部</td><td> 90° - 180° </td><td colspan="2"> electodesのセンターは、ちょうど耳の原基の後方</td><td> E12- E13 </td><td> 1〜1.5μL/ 4から2.7μgの/μL </td><td> 33- 35 </td><td> 0.5〜3ミリメートル</td></tr></tbody></table> 表1：結果のまとめこの表に含まを説明皮質領域の特定のトランスフェクションのためのすべての関連情報です。これは、標的領域、角度、電極の位置、胚段階、DNA溶液、電圧、電極の大きさの塗布量や濃度についての詳細が含まれています。

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

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

27.9K 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

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol we outline the experimental methodology for preparing mice for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E13-E16 mice, however, it is most commonly performed at E15, as shown in this video. 

E15マウス胚の子宮内での脳室内注入とエレクトロポレーションで

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

生物学

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 ...

神経科学

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 ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

によって胚および成体神経幹細胞の拡大<em&gt;子宮内</em&gt;エレクトロポレーションまたはウイルス定位注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

マウス網膜神経節細胞における遺伝子発現のための子宮内および生体外でのエレクトロポレーションで

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

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 ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮質介在ニューロンのサブタイプ選択的エレクトロポレーション

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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, ...

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

時間的および空間的に分離された細胞集団を標的とする子宮エレクトロポレーションの二重

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

齧歯類を用いた子宮内エレクトロポレーション法

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...

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

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