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 border="0" cellpadding="0" cellspacing="0">
	<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: 0.5mm / wall thickness: 0.15-0.2mm. Used for insulating gold tiped electrode</td>
		</tr>
		<tr>
			<td>2.5mm square box filament, 4.5mm 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&#34; 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, 0.12mm Teeth; 3.75&#34; Length; .3mm 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

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

最適化された<em&gt;元OVO</em&gt;開発の進んだ段階にニワトリ胚の培養

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

発生生物学

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 ...

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

共培養系における神経結合の形成を評価するための光遺伝学的アプローチ

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &amp;#8212; A High-throughput Screen in ES Cells

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

ES細胞におけるハイスループットスクリーニングを - マウスにおけるストレス抵抗性遺伝子を明らかにフォワード遺伝的アプローチ

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

チック小脳スライスと顆粒細胞前駆体の空間的にターゲットを絞ったエレクトロポレーションの ex vivo培養

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

チキン聴覚器官への遺伝子導入によって、<em&gt;卵内</em&gt;マイクロエレクト​​ロポレーション

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

小説<em&gt;例OVO</emニワトリ胚のシステムで心臓内血行動態を変えるために&gt;バンディングテクニック

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

長期用水中フィルターペーパーサンドイッチ<em&gt;例OVO</emニワトリ初期胚の&gt;タイムラプスイメージング

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR を介した損失関数における小脳顆粒細胞のエレクトロポレーション法による遺伝子導入の子宮内を使用して

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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 ...