Name: in vivo microinjection and electroporation of mouse testis
Uploaded: 2014-08-23T15:00:00.000+00:00
Duration: 8 min 39 s
Description: Watch this Scientific Journal Video about In Vivo Microinjection and Electroporation of Mouse Testis at JoVE.com

私たちは、精巣マイクロインジェクションを教えるためのミュンスター大学で生殖医療アンドロロジーのセンターのビルギットWesternstroeerに感謝します。加えて、私たちは技術支援のためのウルスラAntkewitzとペトラRecklingに感謝しています。私たちは、この作品（WE2458 / 10-1）を支持するためのドイツ研究財団（DFG）をお願いいたします 。 &#160;

すべて行った動物実験はローカル倫理委員会（LandesamtエリーゼLandwirtschaft、LebensmittelsicherheitウントFischerei、メクレンブルク=フォアポンメルン州、ドイツ）によって承認されている。 1プラスミドの準備<ol><li>動物の免疫反応を回避することができるように、プラスミド調製のために、エンドトキシン除去緩衝液を用いてプラスミド精製キット（材料テーブルを参照）、または類似の方法を使用する。マニュアルの指示に従ってください。プラスミド溶液を希釈するためのddH 2 Oを使用する。 </li><li>最高速度（20,000×g）でDNA溶液を紡糸して破片を除去します。その後、上清を回収。 </li><li>分光光度計（資料を参照）、プラスミド濃度を決定する。 </li><li> 1-3μgの（DNA）/μlにのddH 2 Oを有するプラスミド濃度を調整します。注：より高い濃度があまりにも粘性の注射液が発生する可能性がありながら、より低い濃度では、トランスフェクション効率が低下します。加えて、トランスフェクション効率は、プラスミドの大きさに依存し、個別にテストされなければならない。 </li><li>注射の前に、例えばで5μlのPBS（10倍）と5μlのファストグリーン（0.5％）と一緒に40μlのプラスミドソリューションミックスを準備します。ファストグリーンは、注入プロセスを追跡するために必要とされる。好ましくは、薄壁のPCRチューブあるいはパラフィルムを使用しています。注：精巣のサイズに応じて、20〜50μlの容量は、各精巣のために必要とされる。 </li></ol>マイクロインジェクションピペットの作製<ol><li>マイクロインジェクションピペットを調製するためのホウケイ酸毛細血管を（材料の表を参照）を使用します。 </li><li>垂直キャピラリープラーとガラスキャピラリーを引いて（材料の表を参照）。 </li><li>そっとバンディングによって鉗子でキャピラリー先端ブレーク。チップは通常、50〜80ミクロン、直径が約1センチです。これらは、組織を貫通するのに十分な剛性ではないので、長すぎるのヒントを避けるようにしてください。 </li><li>最後、舎で（材料の表を参照）マイクロピペットbevelerを使って、30°〜45°の角度で先端部をrpen。 </li><li>注射液ミックス（プラスミドの準備を参照）、マイクロインジェクションピペットをロードします。小さなシリンジでガラスキャピラリーの背面に解決策を適用します。注：導電率を低下させるだけでなく、場合によっては組織の損傷の原因となりますので、それ以外の場合、これらはプラスミド溶液と一緒に精細管に注入され、ロード中に気泡を避けるようにしてくださいと。 </li></ol> 3麻酔と手術<ol><li>無菌材料（ すなわち、注射器、注射針、手術器具など）を使用して、無菌状態で操作を実行します。これは、動物の感染を低減し、良好な生存率を確保する。 </li><li> 6-8週齢でエレクトロポレーションのための雄マウスを使用してください。 </li><li>術後鎮痛治療を初期化するには、鎮痛薬で、マウスを提供する手術前に日に飲料水を追加しました。このお尻可能性が高い手術後hypodipsia（減少取水）の場合、先制鎮痛をURES。この目的のために、例えば、小児イブプロフェン懸濁液（20 mg / ml）を用いて飲料水を露出させる。薬用飲料水も初日と同一の濃度の回復の2に供給してください。これは、C57BL / 6J歳8週間25グラムの5ミリリットル/ dの飲料水や体重の有効な7.5 mg / kgを一日量を意味する。この鎮痛レジメンは、根本的な痛みの軽減と高福祉26と一緒に加速し、回復を保証します。 </li><li> 1の比率（材料表を参照）。手術当日麻酔作業溶液を調製するために、1で10％ケタミンおよび2％キシラジンを混ぜる。 </li><li>動物を麻酔するためには、0.25ミリリットル/麻酔液を皮下（;キシラジン= 0.025グラム/ kgのケタミン=キロ0.125グラム/）100mgの体重を適用します。マウスの臓器障害や怪我を防ぐために、骨盤および四肢の間に注射部位を使用してください。通常、10分が必要とされているマウスまで深く麻酔をかけている。 </li><li>麻酔中の乾燥を防ぐために、獣医軟膏をマウスの眼をカバー。 </li><li>応答の完全な欠如による顕著です深い麻酔停止を、テストします。そのためには、単に動物のつま先を挟ま。 </li><li>手術を開始するには、電気シェーバーまたは同様に腹部の毛を除去し、steriliumまたは類似した動作領域を消毒。 </li><li>直接腹部領域の中心にある包皮腺上記腹側切開を加えます。その場所で、第離れて皮を引き、約8〜14ミリメートルの小さな横方向の皮膚のカットを実施しています。その後、腹部の筋肉の層に沿って継続する。 注：病変は、動物に害を低減するためにできるだけ小さくすべきである。 </li><li>添付の精巣を露出し、ちょうど切開部位（ 図2A）の横に先立って準備された防水使い捨ての紙のドレープの上に配置するために慎重に腹部の脂肪パッドを引き上げます。 </li><li> a〜fの双眼鏡を使用してください注入の対象となる輸出管をケガニ動脈管の遠心性は、精巣および精巣上体の間に細かい血管の接合部として識別されます。それは、著名な精巣動脈に隣接して配置される。動脈管は、動脈に45°の角度でほぼ動作し、目に見えて頭の精巣上体に入る。注：マウスの​​年齢に応じて、動脈管は通常、脂肪組織に埋め込まれている。 </li><li>この脂肪組織の輸出管をクリアするために細かい鉗子を使用してください。その後、周囲を損なうことなく、ダクトの明確な視認性を確保するために、無菌の紙片にリリース動脈管を配置します。 </li></ol> 4。マイクロインジェクションとDNAの応用<ol><li>マイクロマニピュレーター/インジェクタユニットへのプラスミド溶液がロードされているマイクロインジェクションピペットを、接続してください。 </li><li>精巣網（ 図2B）の方を向いて先端を輸出管へのマイクロインジェクションの針を平行に配置します。 </li><li>細かいピンセットを使用して、マイクロインジェクションピペットを介してダクトを取り除く。それを上に引っ張りながらキャピラリーダクトと平行に保たれていることを確認します。 注：この手順では、マイクロマニピュレーターで針を移動させることにより、容器を貫通するよりも便利です。 </li><li>精巣に向かって慎重に針を向けると、それが白膜（ 図2C）の直下に精巣網を貫通するのと同様に停止します。 注：精巣網を行き過ぎることの場合には、プラスミド溶液は、間質空間へ漏れる。これは、一般的に精細管をトランスフェクションの失敗につながる。 </li><li> PI：100ヘクトパスカル、TI：0.2秒、およびpc：0ヘクトパスカル（ 図2D）プラスミド溶液の注射のために以下の設定でマイクロインジェクターを利用する。 </li><li>緑色の助けを借りて、ステータスを充填精巣を観察することにより、全体の注入プロセスを監視します。精巣にのみ、PLでその体積の3分の2まで充填されていることに注意してくださいasmid溶液（ 図2E）。 注：注入量を超えた場合、精巣組織が悪影響を受ける可能性がある。 </li></ol>精巣の5。エレクトロポレーション<ol><li>効果的なエレクトロポレーションを有効にするには、PBS（1×）でピンセット電極を浸す。これには、十分な導電性を保証します。 </li><li>スムーズに湿った電極（ 図2F）との間の精巣を絞る。エレクトロとの電気抵抗を測定します。 </li><li>精巣に現在適用されます。 50ミリ秒パルス時間と950ミリ秒間隔時間の一定期間を持つ4つの異なる精巣サイトで40 Vの8矩形パルスを実行します。 </li></ol> 6。創傷閉鎖とポスト手術<ol><li>エレクトロポレーションが終了すると、元の場所に精巣を配置します。 </li><li>外科用縫合糸（資料を参照）、内側筋層を縫う。 </li><li>縫合クリップ（材料表を参照）を採用することにより、皮膚を閉じます。肌アップウィットを引いて時間ピンセット。筋層を除外することを確認します。 5mm以下の距離で各クリップを配置します。 注：外科用縫合糸がマウスで飲み込むことができるため、縫合糸クリップは、皮膚病変を閉じるため好まれる。 </li><li>マウスは、37℃暖かいパッド上に鍛えている無菌の空のマウスのケージに入れ、滅菌したペーパータオル上麻酔から回復できるようにします。パッドは、熱勾配を作成するケージの半分の温暖化を確実にする必要があります。動物の場合、これが原因ケージ内の温度さまざまな個人、最も快適なエリアを検索することができます。応力をさらに低減することができるように、また、滅菌ペーパータオルのシートでマウスをカバーする。注：マウスの​​体表面は、体重に関連してまれに高いので、より大きな動物と比較した場合、熱支持体は、げっ歯類の正常な回復のために特に重要である。 </li><li>操作した動物が完全に目覚めたとき、私を転送完全装備新しいマウスケージへのさらなる回復のためのT。古いケージにまたはマウスのグループに戻し、動物を入れないでください。ケージは創傷感染を防ぐために、できるだけ清潔で無菌であることを確認してください。全体回復プロセスを監視します。それは完全に回復し、正常に動作するように表示されるまで、バック動物飼育施設にマウスを転送するには、待ってください。 注：追加per-と手術後の戦略がプリチェットコーニングKR ら 6によって議論されている。 </li></ol>

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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Evaluation+of+Analgesic+Regimens+for+Abdominal+Surgery+in+Mice.">An Evaluation of Analgesic Regimens for Abdominal Surgery in Mice.</a> J Am Assoc Lab Anim Sci. 6, 18-23 (2000).</li></ol>

マーテンミカエリス、アレクサンダーSobczak、そしてヨアヒムM. Weitzelは生殖生物学の研究所で採用、家畜バイオロジーライプニッツ研究所（FBN）は、Dummerstorf、ドイツは、彼らが競合する金融利害がないことを宣言します。

生殖生物学の分野での研究が、特に男性の生殖能力と精子形成の分野で、必然的に生物に依存しています。精巣機能を調べるために、充分な細胞培養/ in vitro系は半数体精子成熟1,2の二倍体精原細胞からのすべての重要な形態学的変化を反映することが可能な確立されていない。このように、遺伝子改変動物の発生がしばしば必要と男性生殖生物学のような貴重な?...

哺乳動物の精子形成を連続して成熟した半数体精子へと発展させるために、有糸分裂、減数分裂および分化を受けている自己再生幹細胞の洗練されたプロセスであると考えられる。これらの形態学的変化は、異なる細胞型によって編成され、深遠な試みにもかかわらず、それは細胞培養1,2に、これらのプロセスを模倣することは不可能である。そのため、今までの精子形成の研究は、in vivoモデルとして生物に依存しています。一般に、遺伝子機能の研究は、通常、トランスジェニック動物に基づいている。しかしながら、動物モデルのこの種を生成し、維持することは時間がかかり、コストがかかり、非常に精巧である。これは、世代にわたって導入遺伝子を生成し、維持するために必要な長い育種プロセスに起因する。グラムをターゲットにするとさらに、トランスジェニックまたはノックアウトアプローチによる生物全体の遺伝子操作は、生理的障害を引き起こす傾向がある複数の地域に不可欠な機能を備えたエン例えば 、精巣の外側または全身。 さらに、いくつかの一過性のトランスフェクション法は、いくつかの重大な欠点に関連している。リポフェクション3および微粒子ボンバードメント4が組織に損傷を与える可能性があり、十分なトランスフェクション効率のための特定のセルの深さに制限されているのに対し、例えば、ウイルス媒介遺伝子導入の典型的な欠点は、免疫反応および追加の安全規制の可能挑発である。 これとは対照的に、一過性トランスフェクションの別の一般的な方法として、エレクトロポレーション（EP）は、 生体内でトランスフェクションおよび一貫性のあるin vivoでの遺伝子解析に有効にするための有望な技術を構成しているようだ。一般的に、EPはナノ秒以内に結果的に媒介される細孔を有するローカル膜貫通電圧に依存する動的な現象と呼ばれている。これらのギャップは、ミリ秒間維持することができ、十分なトンO DNA、RNA、または小分子5へのアクセス権を付与します。印加電圧が高すぎると、EPの通常は一時文字は、熱産生およびセル5の結果として不可逆的な損傷があまりにも包括的透過性の誘導に打ち消される。 ここでは、エレクトロポレーションは、in vivoでの精巣の遺伝子特性を解明するために、遺伝的精巣形質転換のために利用されることができる効果的かつ経済的なトランスフェクション系であることを示している。この記事では、輸出管およびマウス精巣のその後のエレクトロポレーションを介したプラスミド調製、マイクロインジェクションに対応しています。この手順は、精子形成の過程を調べるために、in vivoでのマウス精巣の精細管の、速く特異的かつ効率的なトランスフェクションを達成するための最適な手段となり得る。

<table><tbody><tr><td>Centrifuge</td><td>Sigma</td><td>1-15 PK</td><td></td></tr><tr><td>Spectrophotometer</td><td>Nanodrop</td><td>ND-1000</td><td></td></tr><tr><td>Micropipette puller</td><td>Narishige</td><td>PC-10</td><td>vertical capillary puller</td></tr><tr><td>Microinjection capillaries</td><td>Clark</td><td>GC100-10</td><td>borosilicate standard wall</td></tr><tr><td>Micropipette beveler</td><td>Bachofer</td><td>Typ 462</td><td>rotating disk beveler</td></tr><tr><td>Electroporator</td><td>Nepagene</td><td>CUY 21EDIT</td><td>square wave electroporator</td></tr><tr><td>Tweezer electrodes</td><td>Nepagene</td><td>CUY 650P5</td><td>5 mm &amp;Oslash; disk electrodes</td></tr><tr><td>Stereo microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>Cold light source</td><td>Zeiss</td><td>KL 1500LCD</td><td></td></tr><tr><td>Microinjection pump</td><td>Eppendorf</td><td>Femtojet</td><td></td></tr><tr><td>Micromanipulator</td><td>homemade</td><td></td><td>XYZ cross table</td></tr><tr><td>Surgical instruments</td><td>FST</td><td></td><td>scissors, forceps, needle holder</td></tr><tr><td>Fine forceps</td><td>FST</td><td>Dumont #5, #7</td><td>efferent ducts preparation</td></tr><tr><td>Michel clip applying stapler</td><td>FST</td><td>Michel, 12029-12</td><td></td></tr><tr><td>Michel suture clips</td><td>FST</td><td>Michel, 12040-01</td><td>7.5 x 1.75 mm</td></tr><tr><td>Surgical suture</td><td>Catgut, Markneukirchen, Germany</td><td>Catgut 00</td><td></td></tr><tr><td>Syringe with 30 G needle</td><td>B. Braun</td><td>Omnican 40</td><td>to load micropipette and for anesthesia</td></tr><tr><td>Plasmid isolation kit</td><td>Promega</td><td>Cat. # A2495</td><td>plasmid Midiprep</td></tr><tr><td>Plasmid pEGFP-C1</td><td>Clontech</td><td>Cat. #6084-1</td><td>CMV-promoter + EGFP</td></tr><tr><td>Plasmid pDsRed2-N1</td><td>Clontech</td><td>Cat. #6084-1</td><td>CMV-promoter + DsRed2</td></tr><tr><td>Fast Green dye</td><td>Sigma</td><td>F7258-25G</td><td>for dilution in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>10% Ketamine</td><td>Serum Werk, Bernburg, Germany</td><td>Urotamin</td><td>mix in a rate 1:1 with xylazine</td></tr><tr><td>2% Xylazine</td><td>Serum Werk, Bernburg, Germany</td><td>Xylazin</td><td>mix in a rate 1:1 with ketamine</td></tr><tr><td>Sterilium</td><td>Bode Chemie</td><td>Sterillium</td><td>disinfection</td></tr><tr><td>Vet ointment</td><td>S&amp;amp;K Pharma, GmbH</td><td>Kerato Biciron 5%, Augensalbe</td><td>opthalmic ointment to prevent eye dryness</td></tr><tr><td>To-Pro-3 iodide</td><td>Invitrogen</td><td>T3605</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;10x PBS, pH 7.4&lt;/strong&gt;</td></tr><tr><td>1.37 M NaCl</td><td>Carl Roth</td><td>3957.1</td><td></td></tr><tr><td>Ibuprofen, Dolormin</td><td>Johnson &amp;amp; Johnson Consumer Health Care Germany</td><td>01094902</td><td>analgesic pediatric solution (NSAID) for postsurgery pain relief</td></tr><tr><td>27 mM KCl</td><td>Carl Roth</td><td>6781.1</td><td></td></tr><tr><td>100 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Carl Roth</td><td>T106.2</td><td></td></tr><tr><td>18 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Carl Roth</td><td>3904.1</td><td></td></tr></tbody></table>

in vivo microinjection and electroporation of mouse testis

このビデオでは、物品の寄与はマイクロインジェクションおよびin vivoでのマウス精巣のエレクトロポレーションの包括的な説明を提供します。精巣マウス細胞のこの特定のトランスフェクション技術は、精子形成におけるユニークなプロセスの研究を可能にします。 以下のプロトコルは、プラスミド構築物で精巣マウス細胞のトランスフェクションに焦点を当てています。具体的には、強化緑色蛍光タンパク質（EGFP）および赤色蛍光タンパク質（DsRed2の）を発現するpDsRed2-N1ベクターを発現するレポーターベクターのpEGFP-C1を用いた。両方の符号化されたレポーター遺伝子は、ヒトサイトメガロウイルス前初期プロモーター（CMV）の制御下にあった。 マウスの精巣への遺伝子導入を行うために、レポータープラスミド構築物は、生きているマウスの精巣に注入される。そのために、麻酔した動物の精巣を露出し、マイクロインジェクションの部位を調製する。当社の優先場所注射は、精巣および精巣上体の間に精子の解剖輸送ルートとして、最終的に接続された精巣網で、輸出管である。このように、マイクロインジェクション後の精細管の充填が良好に起因染色DNA溶液の使用を管理および制御される。その色の細管構造によって精巣の十分な充填を観察した後、臓器をエレクトロポレーションされています。これは、精巣細胞へのDNA溶液を転送することができます。インキュベーションの3日後、精巣を除去し、そしてトランスフェクションの成功を示す、緑色または赤色蛍光について顕微鏡下で調べた。 一般的に、このプロトコルは、DNAまたはRNA-を配信するために使用することができるが、するために生きているマウスの精巣に構築（オーバー）を発現またはin vivo遺伝子機能解析に容易に遺伝子をノックダウン。さらに、レポーター構築物又は推定上の遺伝子レグラを研究するのに適しているトーリー要素。このように、エレクトロポレーション技術の主な利点は、低労力と組み合わせて、高速なパフォーマンスだけでなく、代替技術と比較して必要な適度な技術設備とスキルです。

それはプロトコルに従って使用されるように、インビボでマウス精巣のマイクロインジェクションおよびエレクトロポレーションを行うための実験設定を図1に示されている。それが工業的に製造されたマイクロピペットを取得することができるにもかかわらず、われわれは、引っ張って私たち自身のピペットを生成することが好ましい（ 図1A ）と、彼らは私?...

Watch this Scientific Journal Video about In Vivo Microinjection and Electroporation of Mouse Testis at JoVE.com

In Vivo Microinjection and Electroporation of Mouse Testis

この記事では、精子形成のユニークなプロセスを研究するために精巣マウス細胞のトランスフェクション技術としてインビボでマウス精巣のマイクロインジェクションやエレクトロポレーションについて説明します。提示されたプロトコルは、ガラスキャピラリーの準備、輸出管を経由してマイクロインジェクション、エレクトロポレーションによるトランスフェクションの工程を含む。

マウス精巣の生体内マイクロインジェクションやエレクトロポレーションで

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

in-vivo-microinjection-and-electroporation-of-mouse-testis

Research

JoVE Journal

Biology

In Vivo Microinjection and Electroporation of Mouse Testis

27.8K ビュー.  Leibniz Institute for Farm Animal Biology (FBN). この記事では、精子形成のユニークなプロセスを研究するために精巣マウス細胞のトランスフェクション技術としてインビボでマウス精巣のマイクロインジェクションやエレクトロポレーションについて説明します。提示されたプロトコルは、ガラスキャピラリーの準備、輸出管を経由してマイクロインジェクション、エレクトロポレーションに?...

ビデオ: In Vivo Microinjection and Electroporation of Mouse Testis

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

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

In vivo Electroporation of Developing Mouse Retina

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

In vivo Electroporation of Developing Mouse Retina

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

開発マウス網膜の in vivoエレクトロポレーションで

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

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

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

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

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

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

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

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

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用して哺乳類の骨格筋のDNAトランスフェクションインビボでのエレクトロポレーション

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. However, the time and cost associated with colony creation and maintenance are high. Recent advances in CRISPR-mediated genome editing have allowed the generation of somatic GEMMs (S-GEMMs) by directly targeting the cell/tissue/organ of interest.
The oviduct, or fallopian tube in humans, is considered the tissue-of-origin of the most common ovarian cancer, high-grade serous ovarian carcinomas (HGSCs). HGSCs initiate in the region of the fallopian tube distal to the uterus, located adjacent to the ovary, but not the proximal fallopian tube. However, traditional mouse models of HGSC target the entire oviduct, and thus do not recapitulate the human condition. We present a method of DNA, RNA, or ribonucleoprotein (RNP) solution microinjection into the oviduct lumen and in vivo electroporation to target mucosal epithelial cells in restricted regions along the oviduct. There are several advantages of this method for cancer modeling, such as 1) high adaptability in targeting the area/tissue/organ and region of electroporation, 2) high flexibility in targeted cell types (cellular pliancy) when used in combination with specific promoters for Cas9 expression, 3) high flexibility in the number of electroporated cells (relatively low frequency), 4) no specific mouse line is required (immunocompetent disease modeling), 5) high flexibility in gene mutation combination, and 6) possibility of tracking electroporated cells when used in combination with a Cre reporter line. Thus, this cost-effective method recapitulates human cancer initiation.

Somatic Genome-Engineered Mouse Models Using In Vivo Microinjection and Electroporation

This protocol describes microinjection and in vivo electroporation for regionally restricted CRISPR-mediated genome editing in the mouse oviduct.

in vivoマイクロインジェクションとエレクトロポレーションを用いた体細胞ゲノム改変マウスモデル

Germline genetically engineered mouse models (G-GEMMs) have provided valuable insight into in vivo gene function in development, homeostasis, and disease. ...

がん研究

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

マウスでXenografting胚細胞移植と精巣組織

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms of human diseases such as cancer. Transgenic and conditional knockout mice have been frequently used for gene overexpression or ablation in specific tissues or cell types in vivo. However, generating germline mouse models can be time-consuming and costly. Recent advancements in gene editing technologies and the feasibility of delivering DNA plasmids by viral infection have enabled rapid generation of non-germline autochthonous mouse cancer models for several organs. The bladder is an organ that has been difficult for viral vectors to access, due to the presence of a glycosaminoglycan layer covering the urothelium. Here, we describe a novel method developed in lab for efficient delivery of DNA plasmids into the mouse bladder urothelium in vivo. Through intravesical instillation of pCAG-GFP DNA plasmid and electroporation of surgically exposed bladder, we show that the DNA plasmid can be delivered specifically into the bladder urothelial cells for transient expression. Our method provides a fast and convenient way for overexpression and knockdown of genes in the mouse bladder, and can be applied to building GEMMs of bladder cancer and other urological diseases.

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder diseases.

電気穿孔法によるマウス膀胱尿路上皮にプラスミド DNA 配信法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes are critical for the investigations of spindle assembly and chromosome segregation in male meiotic division. The mouse spermatocyte is an ideal model for mechanistic studies of meiosis, however, the effective methods for the analyses of spermatocytes are lacking. In this article, a practical and efficient method for the in vivo inhibition of kinesin-7 CENP-E in mouse spermatocytes is reported. A detailed procedure for testicular injection of a specific inhibitor GSK923295 through abdominal surgery in 3-week-old mice is presented. Furthermore, described here is a series of protocols for tissue collection and fixation, hematoxylin-eosin staining, immunofluorescence, flow cytometry and transmission electron microscopy. Here we present an in vivo inhibition model via abdominal surgery and testicular injection, that could be a powerful technique to study male meiosis. We also demonstrate that CENP-E inhibition results in chromosome misalignment and metaphase arrest in primary spermatocytes during meiosis I. Our in vivo inhibition method will facilitate mechanistic studies of meiosis, serve as a useful method for genetic modifications of male germ lines, and shed a light on future clinical applications.

Functional Assessment of Kinesin-7 CENP-E in Spermatocytes Using In Vivo Inhibition, Immunofluorescence and Flow Cytometry

This article reports an in vivo inhibition of CENP-E through abdominal surgery and testicular injection of GSK923295, a valuable model for male meiotic division. Using the immunofluorescence, flow cytometry and transmission electron microscopy assays, we show that CENP-E inhibition results in chromosome misalignment and genome instability in mouse spermatocytes.

in vivo阻害、免疫蛍光法およびフローサイトメトリーを用いた精母細胞におけるキネシン-7 CENP-Eの機能評価(英語)

In eukaryotes, meiosis is essential for genome stability and genetic diversity in sexual reproduction. Experimental analyses of spermatocytes in testes ...