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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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 border="0" cellpadding="0" cellspacing="3">
	<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 &#216; 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 00</td>
			<td>Catgut 
			Markenkirchen</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe, with 30G 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 ddH2O</td>
		</tr>
		<tr>
			<td>10x PBS, pH 7.4</td>
			<td>Carl Roth</td>
			<td>3957.1</td>
			<td>NaCl [1.37 M]</td>
		</tr>
		<tr>
			<td>6781.1</td>
			<td>KCl [27 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T106.2</td>
			<td>Na2HPO4+2H2O [100 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3904.1</td>
			<td>KH2PO4 [18 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Ketamin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Urotamin</td>
			<td>mix in a rate 1:1</td>
		</tr>
		<tr>
			<td>2% Xylazin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Xylazin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilium</td>
			<td>Bode Chemie</td>
			<td>Sterillium</td>
			<td>disinfection</td>
		</tr>
		<tr>
			<td>vet ointment</td>
			<td>S&#38;K Pharma 
			GmbH</td>
			<td>Kerato Biciron 5% 
			Augensalbe</td>
			<td>or similar to prevent 
			dryness of eyes</td>
		</tr>
		<tr>
			<td>To-Pro-3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</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

27.6K 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

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

無傷の開発脳内の生体内の単一細胞のエレクトロポレーション

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

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 ...

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 ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

シトクロムの細胞質マイクロインジェクションによるアポトーシスの活性化 C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

マウス卵母細胞のマイクロインジェクション、成熟および倍数性評価

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

アダルトゼブラフィッシュ網膜にモルフォの in vivoエレクトロポレーションに

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

リジェネアダルトゼブラフィッシュテールフィンにMorpholinos の in vivoエレクトロポレーションで

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...