Name: inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation
Uploaded: 2017-11-17T15:00:00.000+00:00
Duration: 10 min 29 s
Description: Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

著者らは、ジェームズ ・ マディソン大学生物教室・ ジェームズ ・ マディソン大学光顕微鏡イメージング施設の寛大なサポートをありがとうございます。夫妻ジャスティン ・ w ・ ブラウンとコーリー ・ l ・ クレランド手術材料と空間の寛大な調整のために博士マーク ・ l ・ ガブリエレ若い生後ティッシュの準備、に関する有益な助言を。この研究はだった 4-バージニア州、連邦のバージニア州 (G.S.V.) を推進するための共同パートナーシップとによってバージニア州アカデミーの科学小プロジェクト研究助成金 (G.S.V.) 共同研究助成金によって一部で賄われて。学部研究生奨学金 (K.M.B.) に、ファレル夏研究奨学金 (K.M.B.) をジェームズ ・ マディソン大学第 2 世紀への奨学金 (K.M.B.)、寛大サポート ベティ Jo 愛するバトラー 58年基金によって提供されているジェームズマディソン大学百周年記念奨学金 (C.J.H.) に、ジェームズ ・ マディスン大学ルーシー ロビンソン検索 30年記念奨学金 (Z.L.H.) に、科学と数学教員援助交付金 (G.S.V.) にジェームズ ・ マディソン大学。

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著者はある利益相反を開示します。

モザイク脳組織を生成する floxed マウスにおける Cre リコンビナーゼとエレクトロポレーションの子宮内の組み合わせを紹介します。このアプローチの利点は、新しいマウス行が異なる細胞サブタイプが対象とするたびに生成する必要はありません: 興奮性ニューロン、抑制ニューロンまたは時間によってグリア細胞を対象に子宮内でエレクトロポレーションを使用できますエレ?...

興味の遺伝子の機能を理解するための古典的な実験パラダイムは、遺伝のモザイクを生成します。遺伝子は細胞の表現型に必要なかどうかを決定するには、最も簡単な方法は有機体 (例えばノックアウト) を通して遺伝子の機能の損失を引き起こしています。ただし、遺伝子が特定の細胞型で特に必要なかどうかを決定、生物全体の遺伝子のノックアウトは有効なアプローチではありません。代わりに、メソッドが必要なしの野生型 (すなわち遺伝的摂動) 組織に囲まれている間特定のセルに遺伝子の機能の損失と、-つまり、モザイク組織を作成します。突然変異体の細胞が突然変異体の表現型を示していますが、周囲の野生型細胞は、細胞自律的方法で遺伝子機能。解析モザイク組織、変異細胞が野生型組織に囲まれているは、遺伝子、特にニューロンとグリア細胞が組織の広大な相互接続ネットワークを形成する脳の細胞の自律的機能の理解に最適です。
モザイク脳組織のいくつかのフォームは、遺伝子の細胞の自律的機能を調べるための強力なモデルを提供しています。神経移植1、女性 X-リンクされてモザイク2,3,4に焦点を当てた研究とモザイクに基づいて彼らの結論を描画している内因性の体細胞モザイク5,6脳組織。Cre lox 組換えシステムを介して遺伝子の条件削除は、トランスジェニック マウス線の偉大な可用性を最大限に活用する方法です。このメソッドは、loxP サイト同じ方向 ("floxed") の両方の顔が並ぶそれを残して (エクソン) など遺伝子の必要なシーケンスのいずれかの側で 2 つの loxP サイトを紹介しています。Cre リコンビナーゼ loxP サイト7間のシーケンスを切り取る。Cre を介する組み換えは、セル (「Cre 記者ライン」) のサブセットに蛍光マーカーと一緒に Cre リコンビナーゼを表現する別のマウス線横断 floxed マウスで実現できます。これは、さまざまな興奮性神経細胞やアストロ サイト8などの細胞のサブセットの遺伝子の機能を明らかにする方法で実証されています。Cre 記者行クレエT2 (単一ニューロン誘導 Cre を介したノックアウトまたは滑らかでラベル付け) という薬物誘導する Cre を介した結合を許可するを表すことができます9。ダブル マーカー (MADM)10,11モザイク解析と呼ばれる別の戦略、Cre を介した染色体間組換えホモ接合体変異ヘテロ接合体組織と一緒に作成することができます。これらのアプローチのマウスの新しい行は各候補遺伝子またはテストされる細胞のサブタイプのたびに生成される必要があります。イオントフォレシス12通したり、ウイルスのベクトル (例えばアデノ随伴ウイルス13またはレンチ14細胞サブタイプに固有を運ぶ Cre リコンビナーゼを後天的導入できる代わりに、発起人)。この戦略は、強いと生後のラベルを作成します。まばらと胎生期大脳皮質ニューロンの開発を対象とするには、理想的な戦略は蛍光マーカーで Cre リコンビナーゼのエレクトロポレーション子宮内です。
体内組織モザイク エレクトロポレーション子宮内で生成する Cre lox 再結合を結合することに加えて、その他公開プロトコルから15,16、手順にいくつかの適応を紹介 17,18,19,20,21。繁殖タイミング妊娠した女性の成功を改善するために情報を提供いたします。我々 はまた、大脳皮質組織の神経細胞の疎と明るいラベリングを導入する我々 の 2 つの戦略を概説: Cre リコンビナーゼの蛍光マーカー22single コンストラクト コーディングのレベルを滴定する戦略の 1 つです。別の戦略は、心23,24でこれらのパラメーターを具体的に設計された「超新星」システムを使用することです。さらに、一貫性のあるマイクロインジェクション ピペットと子宮の穿孔手術を簡素化の生産の改善を提供しています。最後に、我々 はさらに汚れるか、または免疫組織化学を樹状突起スパインの樹状突起と軸索のアーバー、分析できる簡易組織の準備で重要な手順を概説します。

<table><tbody><tr><td>C57BL/6J mice</td><td>The Jackson Laboratory</td><td>#000664</td><td>See &quot;1. Mouse set-up&quot; (step 1.1, &quot;wildtype mice&quot;)</td></tr><tr><td>GFP.Cre empty vector</td><td>AddGene</td><td>#20781</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;single DNA construct that codes for Cre recombinase as well as a fluorescent marker&quot;). GFP.Cre empty vector was a gift from Tyler Jacks.</td></tr><tr><td>pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#69138</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK029.CAG-loxP-stop-loxP-RFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK031.TRE-Cre (Supernova)</td><td>AddGene</td><td>#69136</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK031.TRE-Cre (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova)</td><td>AddGene</td><td>#85006</td><td>See &quot;2. DNA set-up&quot; (step 2.1 &quot;Supernova&quot; system) and http://snsupport.webcrow.jp/. pK038.CAG-loxP-stop-loxP-EGFP-ires-tTA-WPRE (Supernova) was a gift from Takuji Iwasato.</td></tr><tr><td>EndoFree Plasmid Maxi Kit (10)</td><td>Qiagen</td><td>#12362</td><td>See &quot;2. DNA set-up&quot; (step 2.3 &quot;endotoxin-free plasmid purification kit&quot;)</td></tr><tr><td>Trypan Blue powder, BioReagent grade</td><td>Sigma</td><td>T6146-5G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;trypan blue&quot;)</td></tr><tr><td>Sodium Chloride, ACS, 2.5 kg</td><td>VWR</td><td>BDH9286-2.5KG</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;NaCl&quot;)</td></tr><tr><td>Potassium Chloride, ACS, 500 g</td><td>VWR</td><td>#97061-566</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KCl&quot;)</td></tr><tr><td>Sodium phosphate dibasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>S0876-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Potassium phosphate monobasic, ReagentPlus, 100 g</td><td>Sigma-Aldrich</td><td>P5379-100G</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&quot;)</td></tr><tr><td>Hydrochloric acid, ACS reagent, 500 mL</td><td>Fisher Scientific</td><td>A144-500</td><td>See &quot;2. DNA set-up&quot; (step 2.5 &quot;HCl&quot;)</td></tr><tr><td>P-97 Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>3.0 mm wide trough filament</td><td>Sutter Instrument</td><td>FT330B</td><td>See &quot;3. Pipette set-up&quot; (step 3.1 &quot;glass capillary puller&quot;)</td></tr><tr><td>Thin Wall Glass Capillaries, 4&quot;, 1 / 0.75 OD/ID</td><td>World Precision Instruments</td><td>TW100-4</td><td>See &quot;3. Pipette set-up&quot; (step 3.1.1 &quot;glass capillary&quot;)</td></tr><tr><td>Single Ply Soft-Tech Wipes, 4.5&quot;</td><td>Phenix</td><td>LW-8148</td><td>See &quot;3. Pipette set-up&quot; (step 3.2.1 &quot;single-ply task wipe&quot;); other single-ply wipes (e.g. Kimwipes) can be used.</td></tr><tr><td>Graefe Forceps, 7 cm, Straight, 0.7 mm 1x2 Teeth</td><td>World Precision Instruments</td><td>#14140</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Graefe forceps&quot;)</td></tr><tr><td>Iris Scissors, 11.5 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503708-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;iris scissors&quot;)</td></tr><tr><td>Hartman Mosquito Forceps, 9 cm, Straight, 12-pack</td><td>World Precision Instruments</td><td>#503728-12</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Hartman mosquito forceps&quot;)</td></tr><tr><td>General Purpose Non-Woven Sponges, 2&quot; x 2&quot;, 4-ply</td><td>Medrepexpress</td><td>#2204-c</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;non-woven gauze sponges&quot;)</td></tr><tr><td>Ring Tipped Forceps, 10 cm, Straight, 2.2mm ID</td><td>World Precision Instruments</td><td>#503203</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;ring-tipped forceps&quot;)</td></tr><tr><td>Pyrex petri dishes complete, O.D. &amp;times; H 100 mm &amp;times; 20 mm</td><td>Sigma-Aldrich</td><td>CLS3160102-12EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;Petri dishes&quot;)</td></tr><tr><td>Flat Type Instrument Tray, Stainless Steel, 13-5/8&quot; x 9-3/4&quot; x 5/8&quot;</td><td>Amazon</td><td>B007SHGAHA</td><td>See &quot;4. In utero electroporation&quot; (step 4.1 &quot;stainless steel tray&quot;)</td></tr><tr><td>Platinum Tweezertrode, 5 mm</td><td>BTX</td><td>#45-0489</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.16 &quot;tweezer-type electrodes&quot;)</td></tr><tr><td>ECM 830 Foot Pedal</td><td>BTX</td><td>#45-0211</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 and 4.17 &quot;foot pedal&quot;)</td></tr><tr><td>ECM 830 Generator</td><td>BTX</td><td>#45-0052</td><td>See &quot;4. In utero electroporation&quot; (step 4.3 &quot;generator&quot;)</td></tr><tr><td>Single Animal Isoflurane Anesthesia System with Small Induction Box</td><td>Harvard Apparatus</td><td>#72-6468</td><td>See &quot;4. In utero electroporation&quot; (step 4.4 and 4.6 &quot;nose cone&quot;, step 4.4 &quot;induction chamber&quot;)</td></tr><tr><td>Ophthalmic ointment</td><td>Hanna Pharmaceutical Supply Co</td><td>#0536108691</td><td>See &quot;4. In utero electroporation&quot; (step 4.7 &quot;veterinary ophthalmic ointment&quot;)</td></tr><tr><td>Space Gel (AIMS)</td><td>VWR</td><td>#95059-640</td><td>See &quot;4. In utero electroporation&quot; (step 4.8 &quot;sealed pouch filled with supersaturated salt solution&quot;)</td></tr><tr><td>Hair Remover Gel Cream, Sensitive Formula</td><td>Veet</td><td>#062200809951</td><td>See &quot;4. In utero electroporation&quot; (step 4.9 &quot;depilatory cream&quot;)</td></tr><tr><td>10ul Low Retention Tip Starter (960 tips/pk)</td><td>Phenix Research Products</td><td>TSP-10LKIT</td><td>See &quot;4. In utero electroporation&quot; (step 4.12 &quot;sterile 10 &amp;micro;L micropipette tip&quot;)</td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma-Aldrich</td><td>A5177</td><td>See &quot;4. In utero electroporation&quot; (step 4.15 &quot;aspirator tube assembly&quot;)</td></tr><tr><td>Braided Absorbable Suture, 4-0, Needle NFS-2(FS-2), 27&quot;</td><td>Medrepexpress</td><td>MV-J397</td><td>See &quot;4. In utero electroporation&quot; (step 4.19 &quot;absorbable sutures&quot;)</td></tr><tr><td>&amp;ldquo;LiquiVet Rapid&amp;rdquo; Tissue Adhesive</td><td>Medrepexpress</td><td>VG3</td><td>See &quot;4. In utero electroporation&quot; (step 4.20 &quot;tissue adhesive&quot;)</td></tr><tr><td>Hypodermic syringes, polypropylene, Luer lock tip, capacity 1.0 mL</td><td>Sigma-Aldrich</td><td>Z551546-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;1 mL syringe&quot;)</td></tr><tr><td>BD Precisionglide syringe needles gauge 26, L 1/2 in.</td><td>Sigma-Aldrich</td><td>Z192392-100EA</td><td>See &quot;4. In utero electroporation&quot; (step 4.21 &quot;26G, &amp;frac12;&amp;rdquo; needle&quot;)</td></tr><tr><td>Nestlets Nesting Material</td><td>Ancare</td><td>NES3600</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;nesting materials&quot;)</td></tr><tr><td>Sunflower Seeds, Black Oil, Sterile</td><td>Bio-Serv</td><td>S5137</td><td>See &quot;4. In utero electroporation&quot; (step 4.24 &quot;sunflower seeds&quot;)</td></tr><tr><td>Paraformaldehyde, 97%</td><td>Alfa Aesar</td><td>A11313</td><td>See &quot;5. Histology&quot; (step 5.1.1 &quot;PFA&quot;)</td></tr><tr><td>Economy Tweezers #3, 11 cm, 0.2 x 0.4 mm tips</td><td>World Precision Instruments</td><td>#501976</td><td>See &quot;5. Histology&quot; (step 5.5 &quot;tweezers&quot;)</td></tr><tr><td>Agar powder</td><td>Alfa Aesar</td><td>#10752</td><td>See &quot;5. Histology&quot; (step 5.8.1 &quot;agar&quot;)</td></tr><tr><td>Single-edge razor blades, #9 blade</td><td>Stanley Tools</td><td>#11-515</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;single-edge razor blade&quot;)</td></tr><tr><td>Specimen disc S D 50 mm</td><td>Leica</td><td>#14046327404</td><td>See &quot;5. Histology&quot; (step 5.9 &quot;vibrating microtome specimen disc&quot;)</td></tr><tr><td>Buffer tray S assembly</td><td>Leica</td><td>#1404630132</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;buffer tray&quot;)</td></tr><tr><td>VT1000 S Vibratome</td><td>Leica</td><td>#14047235612</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;vibrating microtome&quot;)</td></tr><tr><td>Double Edge Razor Blades</td><td>Personna</td><td>BP9020</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;blade&quot;)</td></tr><tr><td>Knife Holder S</td><td>Leica</td><td>#14046230131</td><td>See &quot;5. Histology&quot; (step 5.10 &quot;knife holder&quot;)</td></tr><tr><td>Studio Elements Golden Taklon Short Handle Round Brush Set</td><td>Amazon</td><td>B0089KU6XE</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;fine tipped paintbrush&quot;)</td></tr><tr><td>Superfrost Plus Slides</td><td>Electron Microscopy Services</td><td>#71869-11</td><td>See &quot;5. Histology&quot; (step 5.12.1 &quot;microscope slide&quot;)</td></tr><tr><td>ProLong Diamond Antifade Mountant, 10 ml</td><td>Thermofisher</td><td>P36970</td><td>See &quot;5. Histology&quot; (step 5.12.3-5.12.4 &quot;mountant&quot;)</td></tr><tr><td>Cover Glass, 24 x 50 mm, No. 1</td><td>Phenix Research Products</td><td>MS1415-10</td><td>See &quot;5. Histology&quot; (step 5.12.4 &quot;coverslip&quot;)</td></tr><tr><td>4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Sigma-Aldrich</td><td>D9542</td><td>See &quot;5. Histology&quot; (step 5.13.1 &quot;DAPI&quot;)</td></tr><tr><td>Fixed Stage Upright Microscope</td><td>Olympus</td><td>BX51WI</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;light microscope&quot;)</td></tr><tr><td>Laser Scanning Confocal Microscope</td><td>Nikon</td><td>TE2000/C2si</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;confocal microscope&quot;)</td></tr><tr><td>4x objective, NA = 0.20</td><td>Nikon</td><td>CFI Plan Apo Lambda 4X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;low-power objective&quot;)</td></tr><tr><td>20x objective, NA = 0.75</td><td>Nikon</td><td>CFI Plan Apo Lambda 20X</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;medium-power objective&quot;)</td></tr><tr><td>60x objective, NA = 1.40</td><td>Nikon</td><td>CFI Plan Apo VC 60X Oil</td><td>See &quot;5. Histology&quot; (step 5.15 &quot;high-power objective&quot;)</td></tr></tbody></table>

inducing cre-lox recombination in mouse cerebral cortex through in utero electroporation

遺伝子の細胞自律神経機能の損失を引き起こすことによって明らかになることやニューロンの小さく、まばらな人口の遺伝子の機能を得る。そうニューロンの損失または遺伝子の機能のゲインが遺伝的摂動組織に囲まれているモザイクを生成する必要があります。ここでは、我々 はニューロンの遺伝子の細胞の自律的機能を研究するために使用できるモザイク脳組織を生成するために子宮内でエレクトロポレーションと Cre lox 組換えシステムを組み合わせます。DNA コンストラクト (リポジトリを通じて利用可能)、蛍光ラベルと Cre リコンビナーゼのコーディングが両脇に loxP サイトで子宮を用いたマウス胚の脳における遺伝子を含む皮質ニューロンの開発に導入します。エレクトロポレーション。さらに、子宮内でエレクトロポレーション法の存続性と再現性を向上させる様々 な適応について述べる。このメソッドはまた、ニューロンのスパースまたは密な人口の Cre 介する組み換え価を確立します。ラベル付けされた脳組織の組織学的準備を必要としない (しかしに合わせることができる) の免疫組織化学。使用構造を保証する蛍光ニューロン キャリー Cre リコンビナーゼの遺伝子に分類します。組織学的準備は、アーバーの樹状突起と軸索と樹状突起スパインの共焦点イメージングによるニューロンの形態素解析を許可します。損失または利得関数のスパース モザイク組織で実現するため、このメソッドは、遺伝子製品生体内での細胞の自律的必要性の検討とを許可します。

シングルは、GFP を構築します。Cre は E15.5 で electroporated をされ、P14 の可視化 (材料の一覧を参照してください)。コンストラクトの濃度や注入量、に応じてスパースまたは高密度の結果22,26を取得できます。たとえば、2 mg/mL GFP の 1 μ l 注入。明るい (図 1 a)、および、ローカライズされた層 II/III (<strong...

Watch this Scientific Journal Video about Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation at JoVE.com

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

脳における遺伝子の細胞の自律的機能損失を誘導することによって学ぶことができるまたは細胞のまばらな人口の機能を得る。ここでは、関数の vivoの損失を引き起こす floxed の遺伝子を持つ皮質ニューロンの開発のまばらな人口に Cre リコンビナーゼを提供する子宮内エレクトロポレーションについて述べる。

子宮内エレクトロポレーション マウス大脳皮質における Cre lox 組換えを誘導します。

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

inducing-cre-lox-recombination-mouse-cerebral-cortex-through-utero

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

11.6K ビュー.  James Madison University. 脳における遺伝子の細胞の自律的機能損失を誘導することによって学ぶことができるまたは細胞のまばらな人口の機能を得る。ここでは、関数の vivoの損失を引き起こす floxed の遺伝子を持つ皮質ニューロンの開発のまばらな人口に Cre リコンビナーゼを提供する子宮内エレクトロポレーションについて述べる。

ビデオ: Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific Cre models, deletion of a protein throughout a tissue or cell population often leads to complex phenotypes resulting from multiple interacting mechanisms. Determining whether a phenotype results from disruption of a cell autonomous mechanism, which is intrinsic to the cell in question, or from a non-cell autonomous mechanism, which would result from impairment of that cell&#8217;s environment, can be difficult to discern. To gain insight into protein function in an in vivo context, in utero electroporation (IUE) enables gene deletion in a small subset of cells within the developing cortex or some other selected brain region. IUE can be used to target specific brain areas, including the dorsal telencephalon, medial telencephalon, hippocampus, or ganglionic eminence. This facilitates observation of the consequences of cell autonomous gene deletion in the context of a healthy environment. The goal of this protocol is to show how IUE can be used to analyze a defect in radial migration in a floxed transgenic mouse line, with an emphasis on distinguishing between the cell autonomous and non-cell autonomous effects of protein deletion. By comparing the phenotype resulting from gene deletion within the entire cortex versus IUE-mediated gene deletion in a limited cell population, greater insight into protein function in brain development can be obtained than by using either technique in isolation.

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

タンパク質の削除の誘導を介して<em&gt;子宮内で</em&gt;エレクトロポレーションは、トランスジェニックモデルにおいて神経細胞の移動の欠損を定義する

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

発生生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

E15マウス胚の子宮内での脳室内注入とエレクトロポレーションで

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

生物学

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

神経科学

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

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

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

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

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

神経形態形成の研究のための方法：<em&gt;エキソビボ</em胎児マウス大脳皮質&gt; RNAiのエレクトロポレーション

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作<em&gt;子宮内で</em&gt;エレクトロポレーション

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

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

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

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

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-、Hippocampus-、Thalamus-、Hypothalamus-、横中隔Nucleus-と線条体固有<em&gt;子宮内</em&gt; C57BL / 6マウスでのエレクトロポレーション

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

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

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

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

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

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

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

多アドレス可能なゲノム積分色(MAGIC)マーカーを皮質マウスアストロサイトに個別化する子宮内電気的

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

子宮内エレクトロポレーションマウス大脳皮質における Cre lox 組換えを誘導します。

要約

さらに動画を探す

この動画の章

タンパク質の削除の誘導を介して

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

使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

神経形態形成の研究のための方法：

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作

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

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

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

多アドレス可能なゲノム積分色(MAGIC)マーカーを皮質マウスアストロサイトに個別化する子宮内電気的

子宮内エレクトロポレーション マウス大脳皮質における Cre lox 組換えを誘導します。

要約

さらに動画を探す

この動画の章

タンパク質の削除の誘導を介して

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

使用した複数のCNS準州への効率的な遺伝子送達子宮内でエレクトロポレーション

マウス子宮内でエレクトロポレーション：制御時空間遺伝子導入

神経形態形成の研究のための方法：

使用して、マウス大脳皮質および海馬における可視化と樹状突起と棘の遺伝子操作

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

子宮内エレクトロポレーションは、神経部分集団の興奮とシングルセルの接続を研究するためのアプローチ

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

多アドレス可能なゲノム積分色(MAGIC)マーカーを皮質マウスアストロサイトに個別化する子宮内電気的

子宮内エレクトロポレーションマウス大脳皮質における Cre lox 組換えを誘導します。