私たちは、もともとリファレンス11の130ページに登場し、マイクロインジェクションピペットの作製図を公開する許可をヒューマナプレスに感謝し、ビデオ撮影のためにラリーDlugasとスティーブン·ウォン、教育コミュニケーションのOHSU部、ビデオのデザインと編集のためのラリーDlugas、アダムM. O &quot;クイン、シニアデザイナー、技術的な回路図を提供するため、カスタムの水平層流フードとLesゴールドスミスを設計するためのトリオン/ Envirco、ビクターMonterroso、MV、MS、PhDは、トム·Chatkupt、DVM、比較医学のOHSU部、指導のための私達の動物のケアのプロトコル、手術手技、および予防的鎮痛療法、マルセル·ペレ-Gentilの、DVM、MS、獣医縫合のテクニックで彼の資料を共有するため、私たちのAdobe Premiere Proのビデオ顕微鏡のコンピュータワークステーションを設計するためのエドワードPorsov、MS、およびリア白と（ポートランド、オレゴン州）キャプションLNSのジョナスヒンクリー。この作品は、難聴とオットの国立研究所からの補助金によって支えられてrのコミュニケーション障害：DC R01 008595とDC R01 008595-04S2（JB）およびP30 DC005983（オレゴン聴覚研究センターコアグラント、ピーター·ガレスピー、主任）。

1。腹開腹<ol><li>ペントバルビタールナトリウム麻酔液（グラム体重あたり7.5μL）の腹腔内注射によって、その胚を胚11.5日（膣栓が検出された日の正午には胚発生の日0.5ですE11.5）にあるダムを麻酔。麻酔液作業;無水エタノールを100μL、65 mg / mLの水溶液を硫酸マグネシウム（子宮のトーンを調節する）の320μL、50 mg / mLのペントバルビタールナトリウム溶液18​​0μLおよびプロピレングリコール400μL（と車両の混和を、水性および有機コンポーネント）。 </li><li>足のスクイズを、テールピンチ、そして頬と鼻毛タッチへの応答を点滅させる：有害刺激テストを実施することにより麻酔の完全性を評価します。角膜に無菌眼軟膏を適用します。 </li><li>恥骨部から鋏で胸郭と細かい刃（オスター＃40ブレード）に腹部の毛を剃る。 70％エタノールで腹部、10％ポビドンヨード（Betadine）、消毒ND 70％エタノール、順番に。場所は、滅菌ドレープの上にマウス腹部面を下にして、2から5分の加熱パッドや温かいプレート（37℃）に設定された。 </li><li>ボール先端はさみで腹側正中の腹部皮膚を切開。 10〜14ミリメートルのための切開を拡張します。 白線 、腹壁の腹側正中線に沿って血管、白の結合組織のバンドを識別します。ボールがはさみをひっくり返して白線を切開して切開10〜14ミリメートルを拡張します。すぐに温め（37℃）乳酸リンゲル液で腹部を灌漑。 </li><li>注入部位に過度の圧力を適用せずにリング鉗子で子宮の二本の角を外部化します。予熱し、乳酸リンゲル液で外部化された子宮角を灌漑。 </li></ol> 2。 Transuterineインジェクション<ol><li> 12月16日μ：次のような特性を持つ厚肉、ホウケイ酸ガラスキャピラリーマイクロインジェクションピペットを作製メートルの外径と20度のベベル。 、圧力= 200;速度= 46; = 0を引く時間= 100）熱=ランプのテストに加えて3台：小さなボックスフィラメントとサッターP-97ピペットプラーで、次のプログラムを使用します。サッターBV-10 bevelerで、大口径のピペット用104C（金）の研磨ディスクを使用します。カルシウム遊離リン酸で3-4μg/μLのプラスミドで再懸濁し式は液（pH 7.4）緩衝生理食塩水。濃縮されたDNAは、30秒間穏やかにボルテックスし、10秒、10,000 gでスピンに結晶の高速グリーンを追加します。視覚的に注射の有効性を追跡するために要求される高速グリーンの最小量は、経験的に決定されています。 DNA /ファストグリーンソリューションを斜め​​ピペットのバックフィル。圧力インジェクタ（Picospritzerは、ソースガスとして、&gt; 99％純粋な窒素を使用して）ピペットホルダーにロードされたインジェクションピペットを接続します。 </li><li>低強度は、その注入サイト内の胚を視覚化するためのハロゲンライトで子宮を光を通過させる。 BEAを識別するティン心臓、脳胞、肢芽、後脳の発生期の第 4脳室、アイ。子宮を温めておいで2分毎に灌漑、水和を維持するためにリンゲル液を乳酸。胚の方向、位置を変更すると上記のように解剖学的ランドマークを識別するために、子宮にやさしい圧力を適用します。 </li><li>東洋の前部と後部枝脇腹耳胞が存在する間葉系領域のプライマリ頭静脈を識別するための胚。適切な耳胞は、子宮の透視によって見ることができません。耳胞は、静脈の主幹と一緒に、アメリカンフットボールのフィールド上に支柱またはゴールポストの形状を形成し、主頭静脈の前部と後部の枝の間の中間に位置しています。耳胞は、支柱の間に中間です。 </li><li>耳胞の推定位置と線の軌跡で子宮を介して注入ピペットを挿入します。 uterを通過した後、一度インジェクターパルスを発生私達は、トレーサー色素およびピペットチップのおおよその位置を可視化する。深さを評価するために、再びマイクロ制御とパルスの下にピペットを進めています。さらに繰り返して外側頭の間充織とパルスにピペットを進めています。成功した耳胞のターゲティングは、背側リンパ管と前庭のホタテ貝殻形状のテーパー形状を明らかにする。子宮に圧力を解放し、1モーションで胚/子宮からピペットを削除し、すぐに温めて子宮を灌漑、リンゲル液は乳酸。 </li></ol> （3）in vivoでのエレクトロポレーション<ol><li>乳酸リンゲル液で子宮を灌漑。新たに適用された乳酸リンゲル液は、電気的にカップルパドルスタイルの電極に子宮に必要である。乳酸リンゲルと電極のタングステンの表面を湿らせます。電極のパスの中心に注入耳胞。静かにエレクトロポレーションpaで子宮を圧迫ddles。カソードは、注入された耳胞に横方向の子宮壁に接触しているとアノードuninjected耳胞に隣接した子宮壁と接触している。エレクトロでフットペダルスイッチ付方形波パルス列をトリガします。エレクトロポレーションパラメータは次のとおりです。パルスあたりの43ボルト、950ミリ秒のパルス間の遅延で5、50ミリ秒のパルス。パルスが配信された直後に子宮を灌漑。組織に供給される電流を記録する：60から100 mAmpsは、耳の上皮前駆細胞をトランスフェクトするのに十分である。ダムごとに4-6、E11.5の胚を注入し、エレクトロポ。 </li><li>内側の耳のあるそれらの胚の第 4脳室への水性蛍光デキストラン（発現プラスミドは、緑色蛍光タンパク質をコードしている場合のAlexa Fluor 488発現プラスミドは、赤色蛍光タンパク質またはアレックスFluor 594のをエンコードしている場合）の第二の、独立したtransuterineマイクロインジェクションを実行します。正確に注入し、パルス当たりの電流の少なくとも60 mAmpsは事実であったエレクトロポレーション中ンプのほう。蛍光デキストランは、後脳では出生時に検出でき、内側の耳（3.5を参照）胚発生時に操作された仔の選択が可能になります。 </li><li>乳酸リンゲルと子宮角を灌漑。腹腔内に子宮角を挿入します。予熱し、乳酸リンゲル液2-4 mLで子宮腔をフラッシュし、オーバーフローが滅菌ドレープに切開部位の外に排出することができます。乾燥、滅菌材料とドレープを交換してください。非切削針と6から0吸収性縫合糸で腹壁を縫合。我々は腹壁と皮膚の両方について、他のすべてのステッチをロックして、ランニングステッチを好む。 </li><li>ダムの毛皮を乾燥させ、皮下注射により非ステロイド性抗炎症などのメロキシカムとして管理することができます。滅菌ドレープで温め回復ケージにダムを返します。ダム &#39;呼吸、切開部位の開存性と、流血の放電のために膣を監視し、記録します。出血は、RAである彼女は全身麻酔下にある間、再が存在し、衰えない場合には、ダムを安楽死させる。彼女は意識と歩き回るしようとする試みを取り戻す時間に注意してください。彼女は食べたり飲んだりの兆候を示しており、巣を構築し始めたときにマウスの植民地にダムを返します。通常、これは12時間以内に発生します。 </li><li>出生時（生後0日目）、フラッシュGFPまたはテキサスレッドフィルタでstereofluorescence解剖顕微鏡を用いた蛍光デキストランを検出するごみの各子犬の後脳領域は、必要に応じて設定します。後脳のラベルを表示し、それらの仔だけ授乳中のダムに戻ります。 </li></ol> 4。代表的な結果<img src="/files/ftp_upload/3653/3653fig1.jpg" alt="図1" /> 図1。開発蝸牛へエレクトロポレーションによる遺伝子導入。E11.5耳胞をコードする発現プラスミド強化緑色蛍光タンパク質（EGFP）とエレクトロポレーション（パルスTRAを注入したパラメータ：5、50ミリ秒/パルス、950ミリ秒のパルス間の遅延）で43ボルトのパルス。耳胞蝸牛の中間ターンを介して基地からEGFP発現を示すE11.5で注入し、エレクトロポレーションされた子犬生後6日目（P6）からA）代表的な内耳。蝸牛の外側壁は、中間ターンの頂点のみから削除されました。頂点を生じさせるE11.5前駆細胞はトランスフェクションしていませんでした。 B）で蝸牛の免疫染色ホールマウント（A）は、有毛細胞のマーカーで、ミオシン7aの（MYO7A）は、そのEGFPの発現はコルチ器官の軌跡をたどり、著しく髪細胞有利子感覚上皮に局在していることを示します。耳胞E11.5で注入し、エレクトロポレートしたE18.5胚からC）代表的な内耳。レーザー共焦点投影MYO7A陽性感覚毛細胞のEGFP発現を示しています。 EGFP expressioを示すコルチE18.5臓器から蝸牛感覚上皮のD）レーザー共焦点顕微鏡投影nの内側の有毛細胞（IHC）、外有毛細胞（OHC）、インナーphalangial細胞（IPC）、柱細胞（PC）、およびダイテルス &#39;細胞（DC）であった。 （B）中のスケールバーは（）に適用されます。

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開発マウス内耳への遺伝子導入：マウス内耳は、着床後の開発12,13の最初の週の間に耳プラコードから発生する。胎生9.5（E9.5）で、プラコードは陥入しており、耳胞2と呼ばれる液体で満たされた小胞に変身。小胞の耳前駆体は、成熟した内耳など前庭と聴覚感覚上皮に機械的に敏感な有毛細胞を支配するニューロン内の感覚と非感覚細胞を生じさせる。後期胚?...

<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 </td></tr><tr><td>マイクロ滅菌ケース</td><td> ROBOZ </td><td> RS-9900a </td><td> 8X8.5X1.25インチ</td></tr><tr><td>ボール先端がはさみ</td><td>ファイン科学ツール</td><td> 14109から09 </td><td></td></tr><tr><td>リング鉗子</td><td>ファイン科学ツール</td><td> 11106から09 </td><td> 4.8ミリメートルOD ID/6mm </td></tr><tr><td>アドソンティッシュ鉗子</td><td>ファイン科学ツール</td><td> 11027から12 </td><td></td></tr><tr><td>針ドライバ</td><td>ファイン科学ツール</td><td> 12502から12 </td><td></td></tr><tr><td>アレルギーシリンジトレイ</td><td>ベクトン·ディクソン</td><td> 305536 </td><td></td></tr><tr><td>縫合6から0 </td><td> Syneture </td><td> GL-889 </td><td> 0.7メトリック消化管縫合</td></tr><tr><td>乳酸リンゲル注射液USP </td><td>バクスター</td><td> 2B2323 </td><td></td></tr><tr><td>速いグリーン</td><td>シグマアルドリッチ</td><td> F7258 </td><td></td></tr><tr><td>ホウケイ酸ガラスは、キャピラリ</td><td>ハーバード装置</td><td> 30から0053 </td><td></td></tr><tr><td>ネンブタールナトリウム溶液</td><td> OVATION製薬株式会社</td><td> NDC 67386-501-52 </td><td></td></tr><tr><td> MgSO4.7H2O </td><td>フィッシャー·サイエンティフィック</td><td> M63-500 </td><td></td></tr><tr><td>プロピレングリコール</td><td>フィッシャー·サイエンティフィック</td><td> P355-1 </td><td></td></tr><tr><td>エタノール</td><td>シグマアルドリッチ</td><td> E7023-500 </td><td></td></tr><tr><td>メロキシカム</td><td>ベーリンガーIngeheim </td><td> NADA 141から219 </td><td></td></tr><tr><td>マイクロピペットプラー</td><td>サッター·インスツルメンツ</td><td> P-97 </td><td> FB255Bボックスのフィラメント、サッター機器からピペットクックブックを参照してください</td></tr><tr><td>微小電極Beveler </td><td>サッター·インスツルメンツ</td><td> BV-10 </td><td>大ピペット用104Cベベルディスク、理論をベベルの取扱説明書を参照してください</td></tr><tr><td>マイクロピペットホルダー</td><td>ワーナー·インスツルメンツ</td><td> MP-S15T </td><td> Picospritzerチューブ用外径1.5ミリメートルのピペットとメス圧力ポートのために。 </td></tr><tr><td>ピンセットスタイルの電極</td><td>テックインターナショナル株式会社</td><td> CUY650P5 </td><td> 5ミリメートル、外径</td></tr><tr><td>方形波エレクトロ</td><td>テックインターナショナル株式会社</td><td> CUY21EDIT </td><td>フットペダルを推奨</td></tr><tr><td> PICOSPRITZER III </td><td>パーカー·ハネフィン</td><td> 051-0500-900 </td><td>フットペダルを推奨</td></tr><tr><td>手動制御マニピュレーター</td><td>ハーバード装置</td><td> 640056 </td><td></td></tr><tr><td>水平層流クリーンベンチ</td><td> Envirco </td><td></td><td> LF 630から10554へのカスタム変更。フードの回路図の補足情報を参照してください。 </td></tr><tr><td>ライカstereofluorescenceはLumencor SOLA光エンジンとmicrocopeを解剖</td><td>バーテルズとスタウトとLumencor </td><td> Lumencor SOLA光エンジンとMZ10F </td><td> MZ10Fを集中するとSOLAのライトエンジンをトリガするためにFootpedalsが推奨されます</td></tr><tr><td> Alexa Fluor 594のデキストラン</td><td>インビトロジェン</td><td> D22913 </td><td> 10mg/ml、水性</td></tr><tr><td>のAlexa Fluor®488デキストラン</td><td>インビトロジェン</td><td> D22910 </td><td> 10mg/ml、水性</td></tr><tr><td>エンバイロ-DRI </td><td>シェパード専門診ERS </td><td></td><td> <a href="http://www.ssponline.com" target="_blank">www.ssponline.com</a> </td></tr></tbody></table>

gene transfer to the developing mouse inner ear by in vivo electroporation

、卵形嚢と球形嚢の斑およびコイル蝸牛のコルチ器官半規管の膨大部の3クリステ：哺乳類の内耳は、6異なる感覚上皮を持っています。コルチ器官における聴覚有毛細胞は、1を聞くための主要なトランスデューサている間。クリステと斑は、バランスの特別な感覚を補助するために機械的刺激を伝達する前庭有毛細胞が含まれてい三半規管と蝸牛のこれらの感覚上皮および形態形成における細胞運命の仕様では、マウスの妊娠第2週に行われ、大部分は出生2,3前に完了されています。マウス内耳の発達の研究は、日常的に4,5の細胞および/ ​​または形態学的表現型の分子的基盤への洞察を得るために別の胚または出生後の段階でトランスジェニック胚を採取して実施しています。我々は子宮内での開発、マウス内耳にその遺伝子伝達を仮定する&lt;/ em&gt;は、利得と損失の機能研究の文脈で哺乳類の内耳の開発6の根底にある遺伝的メカニズムの尋問のための伝統的なマウスの遺伝子組換えへの無料アプローチを表しています。 開発マウス内耳における遺伝子強制発現研究を実施するための実験パラダイムは、ここで示した3つの一般的な手順に解決される：1）腹開腹2）transuterineマイクロインジェクション、3） 生体内エレクトロポレーションインチ腹開腹術は、移植胚は7〜実験的なアクセスを得るために子宮の外部化を可能にするマウスの生存率手術手技である。 Transuterineマイクロインジェクションは、耳胞や耳胞の内腔に発現プラスミドを導入するための面取り、ガラスキャピラリーマイクロピペットを使用することである。in vivoでのエレクトロポレーションでは、前駆細胞の8-10に発現プラスミドを駆動するために方形波、直流パルスのアプリケーションです。 11の各ステップで詳細なメモが含まれています。マウスの実験発生学的テクニックが散文から学び、静止画だけでは困難な場合があります。本研究では、我々は、遺伝子導入の手順の3つのステップを示しています。最も批判的に、我々は正確にどのように表示するためにデジタルビデオ顕微鏡をデプロイします。1） 子宮内で胎児の向きを識別する、2）耳胞に注射を標的とするための胚を再設定する、3）耳胞にトレーサー染料溶液と混合し、DNAを顕微注入胚の日11.5と12.5 4）注入された耳胞をエレクトロポ、5）ラベルは、出生時に出生後の選択のための胚をエレクトロポレーション。一般的な方法論的なエラーを回避する方法について説明;耳胞mistargetingの最も一般的な原因に絵のガイド、私たちは正常にトランスフェクトし、内側の耳の代表的な例を提供し、 子宮 g で書くための本ガイドラインエン転送動物ケアのプロトコル。

Watch this Scientific Journal Video about Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation at JoVE.com

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

マウス内耳は、その発達プログラム妊娠中に詳述されているプラ​​コード由来の感覚器官である。我々は定義され<em&gt;子宮内で</em3ステップで構成される&gt;遺伝子導入技術：マウス腹開腹、transuterineマイクロインジェクションし、<emin vivoでの&gt;</em&gt;エレクトロポレーション。我々は、重要な実験発生学的手法を実証するためにデジタルビデオ顕微鏡を使用しています。

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 ...

gene-transfer-to-developing-mouse-inner-ear-vivo

Research

JoVE Journal

Neuroscience

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

13.9K ビュー.  Oregon Health & Science University. マウス内耳は、その発達プログラム妊娠中に詳述されているプラ​​コード由来の感覚器官である。我々は定義され子宮内で遺伝子導入技術：マウス腹開腹、transuterineマイクロインジェクションし、エレクトロポレーション。我々は、重要な実験発生学的手法を実証するためにデジタルビデオ顕微鏡...

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

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

非心室プラスミド注入し、エレクトロポレーションにより産後のラット脳への遺伝子デリバリー

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

神経科学

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

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

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

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

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 ...

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

フローサイトメトリーを用いてマウスの内耳の組織からのLacZの発現細胞を単離する

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

内耳有毛細胞におけるパッチクランプ記録は、単離されたゼブラフィッシュから

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

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 ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

円窓膜を介してマウスインナーイヤーにウイルスにより媒介遺伝子送達のための手術方法

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...