Name: efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach
Uploaded: 2015-11-02T14:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

We would like to acknowledge David O'Brien for his support with data analysis, and all the members of the Canto-Soler lab for their critical discussions. This work was supported by NIH grants EY004859 and EY022631 (MVCS), Core Grant EY1765, and an unrestricted departmental grant from Research to Prevent Blindness, Inc.

この作業で説明するすべての手順は、ジョンズ・ホプキンス大学の動物実験委員会によって推奨されるガイドラインに従って行いました。 事前に1：楽器の調製、試薬と料理<ol><li>卵の調製：卵のインキュベーターで5日間37.5℃、相対湿度60％で受精白色レグホン鶏の卵をインキュベートします。 注：標準化と胚発生を同期させるための役に立つかもしれ容量ロッキングが、ロッキングインキュベーターは、このプロトコルの結果のために厳密には必要ではないです。 </li><li>プラスミド準備： <ol><li>必要に応じてデザインプラスミドは（RNAi技術1,34を使用することによって例えば）遺伝子の過剰発現またはダウンレギュレーションのために構築します。可能な限り、構築物中のレポーター遺伝子を（例えば、EGFP、RFPまたはのLacZなど）が含まれます。 注：例えば、CMVのようないくつかの一般的に使用されるプロモーターは、時間後に沈黙になることができます。 CAGのpromotER（CMVエンハンサーとチキンβ-アクチンプロモーター）関心35-37の遺伝子の効率的な長期的な表現を提供する、非常に良い代替手段です。 </li><li>無菌PBS中1.5μgの/μlの濃度でのプラスミド溶液を準備します。オプション：事前にプラスミド溶液を調製し、それが凍結された使用可能な状態になるまで保管してください。 注：より高いプラスミド濃度が低いプラスミド濃度はトランスフェクション効率1を下げて使用するのに対し、効率の大幅な増加をもたらさありません。 </li></ol></li><li>電極： <ol><li>アノードの使用のために厚い金（特定の材料/機器の表を参照）電極をひっくり返し。 70％エタノールで拭いてください。 注：金の断熱材が露出して約0.5mmを残して、電極を傾け、電流の漏れを最小限にすることをお勧めします。材料を絶縁するための情報は、特定の材料の表に記載されています。 </li><li> 2.5ミリメートル四方bのうち、カソードを作ります牛フィラメント、（通常はマイクロピペットプラーで使用）幅4.5ミリメートル。鉗子を用いて、正方形のボックスを取り消し、長いストリップにフィラメントをまっすぐ。その後、解剖顕微鏡やガイドとして定規を使って下に、ベースで3 mmの長さの正方形「U」形状にフィラメントを曲げ、反対側のフィラメントの残りの長さ（ 図1で、片側に2ミリメートル高、C - D）。ピンセットを使用して、滅菌するために96％エタノール及び火炎に電極を浸します。そして、電極の長い腕に小さなワニ口クリップで電気リード線を接続します。 </li></ol></li><li>エレクトロポレーションチャンバーのセットアップ： <ol><li>テープを使用して100ミリメートルペトリ皿に、ダウン滅菌1.5ミリリットルマイクロチューブの蓋を切断し、それを貼り付け、平らな側がエレクトロポレーションチャンバーを準備する（ 図1E）。 注：仕事と同じような大きさの井戸の形で他の容器と、しかし、マイクロチューブの蓋は簡単にアクセスし、オートクレーブ可能です。よくひよこの目とカソード電極を収容するのに十分な大きさが、プラスミド溶液の廃棄物になるよう大きくないでなければなりません。 </li></ol></li></ol> 2. 例OVOエレクトロポレーション手順<ol><li>楽器の調製<ol><li>マイクロ解剖ツール滅菌浸漬することにより、（RPEを除去するための卵の殻を開いて胚をすくうための大きな湾曲モロニー鉗子の2ペア、微先端湾曲デュモンピンセットの2ペアを、ボンの1組は、レンズと硝子体を除去するための鉗子を歯） 96％のエタノールと炎でのヒント。 70％エタノールに浸したワイプを使用してクリーンな解剖スコープ。 37℃の水浴中で滅菌したカルシウム・マグネシウムフリーハンクス液（CMF-HBSS）のボトルを温めます。暖かいCMF-HBSSでフル3/4に100ミリメートルの滅菌ペトリ皿を埋めます。 </li><li>別のきれいな解剖顕微鏡下でエレクトロポレーションチャンバーを配置し、プラスミド溶液の120μlの記入。 、エレクトロポレーション装置に電極を接続すること必ずカスタムメイドの電極は、負極に接続されていると金が正極に電極をひっくり返しました。 </li></ol></li><li>胚コレクション： <ol><li> 70％エタノールで湿らせたワイプで殻を拭いて卵をきれいにしてください。文化に汚染を運ぶ避けるために、プロトコルの残りの部分で滅菌手順を維持します。大きな湾曲モロニー鉗子を使用して、慎重にゆっくりと（空気室以上）卵の丸い先端をタップして、把持とシェル部分を引き出して卵の殻に穴を開けます。 </li><li>鉗子の別のペアを有する膜を除去し、（胚を傷つけないように注意して）卵からそれをすくうことによって胚を収集します。 CMF-HBSSで皿にそれを置きます。 注：2.4.4で指定されたエレクトロポレーションのために網膜カップの所望の数を取得するために必要な数の胚を収集</li><li>重要なステップ：ハンバーガーとハミルトン38に従って胚をステージ。 注：最適な効率の胚を取得するにはステージ27（ 図2A）でなければなりません。 </li><li>モロニー鉗子を使用して、断頭により胚を安楽死させます。 </li></ol></li><li>網膜カップの入手先： <ol><li>細かいデュモンピンセットを使用して、慎重に目を摘出して、新しいCMF-HBSS皿に移します。 </li><li>視神経乳頭の近くに、各眼の後方部分から開始し、2つの細かいデュモンピンセットを使用して、神経網膜とRPEの間にピンセットの各対の一方の先端を導入し、慎重にRPEが神経を損傷しないように慎重にされて出て解剖します網膜（ 図2B）。 注：RPEを助ける溶液中のCa 2+およびMg 2+の欠如は、より簡単に神経網膜から切り離します。 </li></ol></li><li>エレクトロポレーション： <ol><li> 15ボルト、50ミリ秒の持続時間、および950ミリ秒間隔の5平方パルスを提供するためにエレクトロポのパラメータを設定します。場所 &#39;U&#39;は、エレクトロポレーションチャンバー内の負極を形（微量蓋フィルプラスミド含有溶液の120μL）とのED。 </li><li>湾曲デュモンピンセットを使用して（押していない、すくうにより）エレクトロポレーションチャンバーに1網膜のカップを転送し、電極の底面と上向きレンズ（に向けて網膜の後方部分で、「U」形状の電極内に配置図2D）。 </li><li>それは次のレンズに、目の前の部分に触れると、エレクトロポレーターのフットペダルは、電気パルスを提供するように使用して陽極を配置します。 </li><li> CMF-HBSSで新しいペトリ皿にエレクトロポレーションした網膜カップを移し、残りの網膜カップのそれぞれにエレクトロポレーション手順を繰り返します。 注：6網膜杯までは、トランスフェクション効率の著しい低下なしに同一のプラスミド溶液をエレクトロポレーションすることができます。 </li><li>ボン歯鉗子でそれらを握り、ゆっくりとトンを介して引っ張ることにより、各エレクトロポ網膜カップのレンズと硝子体を削除します湾曲デュモンピンセットの裏で所定の位置に目を保持しながら、彼は口を瞳孔。 </li><li>標準的な手順を以下のエレクトロポ網膜細胞の解離と文化に進んでください。 注：ひよこ網膜細胞の解離と文化に関する詳細なプロトコルの場合ベルガラとカント・ソレル、2012、追加のファイル4〜21を参照して、このプロトコルでは、細胞は、ポリオルニチンでコーティングした6ウェルプレートまたは35に低密度で播種します。 -mm皿（6×10 5細胞 / 35 mmの直径ウェルまたはディッシュ）。使用した培地は、ペニシリン/グルタミン、10％ウシ胎児血清（FCS）および100μg/ mlのリノール酸-BSA 199培地です。培養物を5％CO 2の加湿37℃インキュベーター中で維持されています。この手順を使用して、5 - 6×10 6個の細胞は、典型的には、各ステージ27エレクトロ目ごとに得ることができます。 注：理想的には、せいぜい3時間は、胚の収集および細胞播種の間を通過する必要があり、良好な細胞生存を確保するためにストレスを最小限に抑えます。この時点で、細胞は、良好な形態学的および分子分化を達成していると生存率はまだ高いので観察し、さらなる分析のための典型的なタイミングは、文化の中で4日後です。 </li></ol></li></ol>

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L., Adler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid-mediated+gene+transfection+into+chick+embryo+retinal+cells+in+ovo+and+in+vitro.">Lipid-mediated gene transfection into chick embryo retinal cells in ovo and in vitro.</a> J Neurosci Methods. 104 (1), 1-8 (2000).</li><li>Werner, M., Madreperla, S., Lieberman, P., Adler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+transfected+genes+by+differentiated,+postmitotic+neurons+and+photoreceptors+in+primary+cell+cultures.">Expression of transfected genes by differentiated, postmitotic neurons and photoreceptors in primary cell cultures.</a> J Neurosci Res. 25 (1), 50-57 (1990).</li><li>Yuan, L., Kawada, M., Havlioglu, N., Tang, H., Wu, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+PRPF31+inhibit+pre-mRNA+splicing+of+rhodopsin+gene+and+cause+apoptosis+of+retinal+cells.">Mutations in PRPF31 inhibit pre-mRNA splicing of rhodopsin gene and cause apoptosis of retinal cells.</a> J Neurosci. 25 (3), 748-757 (2005).</li><li>Hughes, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCAS+vector+system.">The RCAS vector system.</a> Folia Biol (Praha). 50 (3-4), 107-119 (2004).</li><li>Das, R. M., 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=A+robust+system+for+RNA+interference+in+the+chicken+using+a+modified+microRNA+operon.">A robust system for RNA interference in the chicken using a modified microRNA operon.</a> Dev Biol. 294 (2), 554-563 (2006).</li><li>Nguyen, A. T., Dow, A. C., Kupiec-Weglinski, J., Busuttil, R. W., Lipshutz, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene+promoters+for+liver+expression+by+hydrodynamic+gene+transfer.">Evaluation of gene promoters for liver expression by hydrodynamic gene transfer.</a> J Surg Res. 148 (1), 60-66 (2008).</li><li>Xu, L., 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=CMV-beta-actin+promoter+directs+higher+expression+from+an+adeno-associated+viral+vector+in+the+liver+than+the+cytomegalovirus+or+elongation+factor+1+alpha+promoter+and+results+in+therapeutic+levels+of+human+factor+X+in+mice.">CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice.</a> Hum Gene Ther. 12 (5), 563-573 (2001).</li><li>You, L. M., 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=A+hybrid+promoter-containing+vector+for+direct+cloning+and+enhanced+expression+of+PCR-amplified+ORFs+in+mammalian+cells.">A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.</a> Mol Biol Rep. 37 (6), 2757-2765 (2009).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> Dev Dyn. 195 (4), 231-272 (1992).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

このプロトコルの成功のための最も重要なステップは、胚の適切な段階を選択することです。以前の出版物では、胚の段階の範囲は、一般に、インキュベーションまたは胚日の日（ED）によって定義され、これらの培養のために与えられています。したがって、通常、EDに6胚をED 5を使用して同等の結果をもたらすと仮定されます。しかし、我々は、上記のように、ステージ27（ED 5）に、トラ?...

ニワトリ胚の網膜からの解離細胞培養物は、広く生存2-9、10-12分化、神経突起伸長13、などを含む光受容体細胞生物学のさまざまな側面を研究するために使用されています。このシステムの利点は、ルーベン・アドラーと共同研究者によって1980年代に開発され、彼と他のグループ14-20によって完成は、動物モデル21としてひよこの固有の特性に存在します。でも、胚の段階でニワトリ目の大きなサイズは、培養のための材料を大量に提供します。培養物は、胚日（ED）5使用して実行されたときにまた、 - 6網膜を、55 -この動物における光受容体の約86％であるので、それらの前駆細胞の80％が光受容体14,15,18,22,23として区別し、コーン24は 、これらの培養物は、この細胞型に焦点を当てた研究のために特に適しています。 我々は最近、develoを持っていますPEDは、したがって、遺伝missexpression研究1を容易にすることによって、このシステムの有用性を広げ、これらの培養物中の細胞の高効率プラスミドトランスフェクションを可能にする簡単な方法を特徴とします。この技術の開発は、細胞自律的に遺伝子機能の研究を可能にするために、トランスフェクションの許容レベルを提供する方法の科学文献中の空隙から茎。主な神経細胞培養は25,26をトランスフェクトするために悪名高い困難なので、これは一部にはあります。この目的のために以前に利用可能な最も一般的に使用される技術の一部は、3-5％の順に効率をもたらすようなリポフェクション、またはリン酸カルシウム媒介トランスフェクションなどの化学的トランスフェクション法を含めて、かなりの毒性27-32を発揮することができます 。酵素レポーター系とプラスミドの使用は、信号を増幅することによって悪いトランスフェクション効率の問題を回避することができるにもかかわらず、彼らはしないでください細胞特異的な効果を識別し、その結果、全体の代表ではないかもしれない小さな細胞集団に基づいています。ひよこのもう一つの広く使用されている方法は、RCASウイルス感染は、増殖細胞にのみ適用し、このプライマリ網膜培養系33に適していないです。 現在のプロトコルではニワトリ胚の目はステージ27（ED 5）、網膜色素上皮（RPE）が除去され、網膜のカップは、プラスミドを含む溶液で満たされたエレクトロポレーションチャンバ内に配置さで摘出し、使用してエレクトロポレーションされているカスタムメイド標準的な技術21を使用して網膜の解離と培養した電極、。この手順を最適化した後、我々は、生存および分化の特性を損なうことなく、一貫して、培養中の細胞の総数の22％単独感光集団内の25％のオーダーでのトランスフェクション効率を達成することができました文化1。ここでは、この技術の成功と再現性を確保するために、この手順のすべての重要なステップを概説し、詳細なプロトコルを提供します。

<table><tbody><tr><td>ECM 830 Electro Square Porator</td><td>BTX/ Harvard Apparatus</td><td>45-0052</td><td></td></tr><tr><td>Genetrode, L-Shaped, 5 mm Gold Tip</td><td>BTX/ Harvard Apparatus</td><td>45-0115 model 512</td><td>Gold tipped electrode used as anode</td></tr><tr><td>Polyimide Tubing</td><td>Vention Medical</td><td>custom made</td><td>Internal Diameter: 0.5 mm/wall thickness: 0.15 - 0.2 mm. Used for insulating gold tiped electrode</td></tr><tr><td>2.5 mm square box filament, 4.5 mm wide</td><td>Sutter Instrument Company</td><td>FB245B</td><td>Used to make cathode electrode</td></tr><tr><td>HBSS, no calcium, no magnesium, no phenol red</td><td>Gibco - Life Technologies</td><td>14175-095</td><td></td></tr><tr><td>Moloney forceps</td><td>Roboz</td><td>RS-8254</td><td>Serrated; Slight Curve; 4.5&quot; Length</td></tr><tr><td>Dumont tweezers 5/45</td><td>Roboz</td><td>RS-5058</td><td>Pattern #5, 45 Degree Angle; .10 X .06mm Tip Size; 109mm Length</td></tr><tr><td>Bonn micro forceps, 1x2 teeth</td><td>Roboz</td><td>RS-5172</td><td>Tying Platform; 1x2 Teeth, 0.12 mm Teeth; 3.75&quot; Length; 0.3 mm Tip Width</td></tr></tbody></table>

efficient gene transfer in chick retinas for primary cell culture studies: an ex-ovo electroporation approach

ニワトリ胚の網膜由来錐体光受容体に富む文化が網膜神経細胞、特に光受容体の生物学を研究し、世界中の研究者のための不可欠なツールとなっています。それらは容易に薬剤開発のためのハイスループット技術に適合させることができるように、このシステムのアプリケーションは、基本的な研究を越え。しかし、これらの培養物における網膜の光受容体の遺伝子操作は、システムの有用性に重要な制限を装った、非常に困難であることが判明しています。我々は最近開発され、現在利用可能な他のプロトコル1に比べて5倍これらの培養物における網膜細胞のトランスフェクションの速度を増加させるプラスミドのエレクトロポレーション技術OVOの元を検証しています。この方法では、ニワトリ胚の眼は、段階27で摘出され、RPEを除去し、そして網膜のカップは、プラスミド含有溶液中に配置されており、容易に構築お客を用いてエレクトロポレーション電極を作りました。網膜は、次に解離し、標準的な手順を用いて培養されます。この技術は、一般的に感光体集団の25％において導入遺伝子の発現を達成する、プラスミドドリブンRNAi技術の使用を介して、例えば、過剰発現の研究​​に、ならびに遺伝子発現のダウンレギュレーションにも適用することができます。現在の出版物のビデオフォーマットは、一次網膜培養における遺伝子機能の研究を可能にする、分野の研究者にこの技術が簡単にアクセスできるようになります。また、成功した結果と再現性のために、この手順の重要なステップの詳細な説明が含まれています。

ここでは、その後の解離細胞培養のための除核ひよこ網膜カップへのプラスミドのトランスフェクションのための単純なプロトコルを提示します。トランスフェクションは、カスタムの電極（ - 2、図1）を作成することが容易に使用してエレクトロポレーションにより達成されます。このプロトコルで説明するパラメータ（平均22％で）20％の間と27の範囲のトランスフェクション...

Watch this Scientific Journal Video about Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach at JoVE.com

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

ニワトリ胚網膜細胞培養物は、感光体の生物学の研究のための貴重なツールを構成しています。私たちは文化の前に網膜のプラスミドエレクトロポOVOの元に基づいて、効率的な遺伝子導入技術を開発しました。この技術はかなりこのシステムの実現可能な遺伝子操作を行う、現在利用可能なプロトコルを介してトランスフェクション効率を向上させます。

初代細胞培養研究のためのひよこ網膜における効率的な遺伝子導入：<em&gt;元オボ</em&gt;エレクトロポレーションアプローチ

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

efficient-gene-transfer-chick-retinas-for-primary-cell-culture

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

8.1K ビュー.  The Johns Hopkins University School of Medicine. ニワトリ胚網膜細胞培養物は、感光体の生物学の研究のための貴重なツールを構成しています。私たちは文化の前に網膜のプラスミドエレクトロポOVOの元に基づいて、効率的な遺伝子導入技術を開発しました。この技術はかなりこのシステムの実現可能な遺伝子操作を行う、現在利用可能なプロトコルを介してトランスフェクション効率を向上さ?...

ビデオ: Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

発生生物学

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental manipulations in ovo/in vivo makes the chicken embryonic retina a versatile and very efficient experimental model. Although the chicken retina is easy to target in ovo by intraocular injections or electroporation, the effective and exact concentration of the reagents within the retina may be difficult to fully control. This may be due to variations of the exact injection site, leakage from the eye or uneven diffusion of the substances. Furthermore, the frequency of malformations and mortality after invasive manipulations such as electroporation is rather high.
This protocol describes an ex ovo technique for culturing whole retinal explants from chicken embryos and provides a method for controlled exposure of the retina to reagents. The protocol describes how to dissect, experimentally manipulate, and culture whole retinal explants from chicken embryos. The explants can be cultured for approximately 24 hr and be subjected to different manipulations such as electroporation. The major advantages are that the experiment is not dependent on the survival of the embryo and that the concentration of the introduced reagent can be varied and controlled in order to determine and optimize the effective concentration. Furthermore, the technique is rapid, cheap and together with its high experimental success rate, it ensures reproducible results. It should be emphasized that it serves as an excellent complement to experiments performed in ovo.

This protocol describes a method to dissect, experimentally manipulate and culture whole retinal explants from chicken embryos. The explant cultures are useful when high success rate, efficacy and reproducibility are needed to test the effects of plasmids for electroporation and/or reagent substances, i.e., enzymatic inhibitors.

エレクトロポレーションおよび化学試薬の治療のための鶏胚からの全網膜の外植片

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

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 ...

神経科学

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

ドーパミン作動性ニューロンの開発における遺伝子機能を研究するためのニワトリ中脳でのOVOエレクトロポレーションに

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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 ...

生物学

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

発展途上ひよこ視蓋接線細胞移動の可視化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as the hair cell. Basic research in the inner ear has led to a deep understanding of how the hair cell functions, and how dysregulation can lead to hearing loss and vertigo. For this research, the mouse has been the pre-eminent model system. However, mice, like all mammals, have lost the ability to replace hair cells. Thus, when trying to understand cellular therapies for restoring inner ear function, complementary studies in other vertebrate species could provide further insights. The auditory epithelium of birds, the basilar papilla (BP), is a sheet of epithelium composed of mechanosensory hair cells (HCs) intercalated by supporting cells (SCs). Although the anatomical architecture of the basilar papilla and the mammalian cochlea differ, the molecular mechanisms of inner ear development and hearing are similar. This makes the basilar papilla a useful system for not only comparative studies but also to understand regeneration. Here, we describe dissection and manipulation techniques for the chicken inner ear. The technique shows genetic and small molecule inhibition methods, which offer a potent tool for studying the molecular mechanisms of inner ear development. In this paper, we discuss in ovo electroporation techniques to genetically perturb the basilar papilla using CRIPSR-Cas9 deletions, followed by dissection of the basilar papilla. We also demonstrate the BP organ culture and optimal use of culture matrices, to observe the development of the epithelium and the hair cells.

In Ovo and Ex Ovo Methods to Study Avian Inner Ear Development

The chick is a cost-effective, accessible, and widely available model organism for a variety of studies. Here, a series of protocols is detailed to understand the molecular mechanisms underlying avian inner ear development and regeneration.

鳥類の内耳の発達を研究するためのOvoおよびEx Ovoの方法

The inner ear perceives sound and maintains balance using the cochlea and vestibule. It does this by using a dedicated mechanosensory cell type known as ...

In Ovo Electroporation in Embryonic Chick Retina

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is comparatively very easy to manipulate the chick embryonic retina using recombinant DNA/RNA technology. Electroporation of DNA/RNA constructs into the embryonic retina have a great advantage to study gene regulation in retinal stem/progenitor cells during retinal development. Different type of assays such as reporter gene assay, gene over-expression, gene knock down (shRNA) etc. can be performed using the electroporation technique. This video demonstrates targeted retinal injection and in ovo electroporation into the embryonic chick retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). Here we show a rapid and convenient in ovo electroporation technique whereby a plasmid DNA that expresses green fluorescent protein (GFP) as a marker is directly delivered into the chick embryonic subretinal space and followed by electric pulses to facilitate DNA uptake by retinal stem/progenitor cells. The new method of retinal injection and electroporation at E4 allows the visualization of all retinal cell types, including the late-born neurons1, which has been difficult with the conventional method of injection and electroporation at E1.52.

In Ovo Electroporation in Embryonic Chick Retina

The overall goal of this video is to show how to perform targeted retinal injection and in ovo electroporation of DNA/RNA constructs into the chick embryonic retina at the Hamburger and Hamilton stage 22-23, which is about embryonic day 4 (E4). This technique is very useful to study gene expression, gene regulation, and morphological change in developing chick retina.

ニワトリ胚の網膜におけるOVOエレクトロポレーションに

Chicken embryonic retina is an excellent tool to study retinal development in higher vertebrates. Because of large size and external development, it is ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

して、古いニワトリ胚への遺伝子導入<em&gt; EX OVO</em&gt;エレクトロポレーション

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Loss-of-Function Approach in the Embryonic Chick Retina by Using Tol2 Transposon-Mediated Transgenic Expression of Artificial microRNAs

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and accessibility for visualization and experimental manipulations. However, its major technical limitation had been the lack of robust loss-of-function approaches for gene function analyses. This protocol describes a methodology of gene silencing in the developing chick retina that involves transgenic expression of artificial microRNAs (miRNAs) by using the Tol2 transposon system. In this approach, a Tol2 transposon plasmid that contains an expression cassette for the EmGFP (emerald green fluorescent protein) marker and artificial pre-miRNA sequences against a target gene is introduced into the embryonic chick retina with a Tol2 transposase expression construct by in ovo electroporation. In the transfected retinal cells, the transposase catalyzes the excision of the expression cassette from the transposon vector and its integration into host chromosomes, leading to the stable expression of miRNAs and the EmGFP protein. In our previous study, we have demonstrated that the expression of Nel, a glycoprotein that exerts multiple functions in neural development, can be significantly suppressed in the developing chick retina by using this technique. Our results indicate that this methodology induces a stable and robust suppression of gene expression and thus provides an efficient loss-of-function approach for studies of retinal development.

We have developed a novel loss-of-function approach that involves the introduction and genomic integration of artificial micro-RNA sequences into chick embryos by using in ovo electroporation and the Tol2 transposon system. This technique provides a robust and stable gene knockdown methodology for studies of gene function during development.

Tol2トランスポゾンを介した人工マイクロRNAのトランスジェニック発現を用いたニワトリ胚網膜における機能喪失アプローチ

The chick retina has long been an important model system in developmental neurobiology, with advantages including its large size, rapid development, and ...

初代細胞培養研究のためのひよこ網膜における効率的な遺伝子導入：

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