我々は彼のHDカメラの使用（キャノンVIXIA HFS100）のために氏ヤミンエンベルに感謝します。この作業は脊髄研究（08から3074-SCR-E-0と10から3091-SCR-E-0）、ブッシュ医学研究賞にN​​IH（EY018738）、ニュージャージー州委員会からの助成金によって部分的にサポートされていました。

以前にマイナーな変更で他のJoveのビデオ2、論文3に記載のプロトコルのいくつかの部分が実行されているので、ここでは詳細にそれらについては説明しません。 1。卵処理と針の準備<ol><li>卵は、最大1週間に約13°Cの時にクーラーワインに格納することができます。温度が高すぎる場合、より低い温度は、高い死亡率を引き起こす一方で、胚は、異常に開発を開始します。一度ワインクーラーから卵を取り出して、大きい方の端に垂直に卵を設定してインキュベートする準備が整いました。 37.5℃のインキュベーターに入れ前に、少なくとも2時間室温で卵を保持します。 </li><li>ハンバーガーとハミルトンステージ22 4から5である胚を取得するために胚の4日目は約96から100時間の卵をインキュベートします。 </li><li>引っ張らガラスキャピラリーチューブからマイクロピペットの針を準備し、解剖マイクロ下に先端を破る直径と20mmのテーパーは0.1μm程度の先端の開口部を得るためにピンセットを持つスコープ。大きなヒントと針が困難な小規模のヒントはDNA溶液をロードして提供する難しさを持っていながら、卵黄膜を貫通しています。 </li><li>マイクロマニピュレータに取り付けられた0.1ミリリットルハミルトンガス密閉シリンジに針を取り付けます。注射器に針を取り付けるためのMasterplexシリコンチューブの小片を使用しています。接続がタイトなこの添付ファイルを密封するためにミネラルオイルを追加する必要はありませんので。 </li><li> 3から6μg/μLのとパラフィルム片の高速グリーン（0.025％）0.2μlの範囲の濃度でレポータープラスミドDNA溶液2μlを混ぜます。ファストグリーン色素が注入を視覚化するのに役立ちます。徐々に、混合物で針をロードします。 </li><li>内膜から卵黄膜を解放するために約180そっと卵を°回転し、元の位置に戻ってそれを回転させ、エレクトロポレーションのためにそれをセットして数分待つ。 </li><li>ゲストフォースドバイを拭く胚への感染を避けるために、70％エタノールでEPSと卵の殻。 </li><li>すぐにサイズのAAピンセットのペアを持つ空気電池上記の卵に小さな穴を作成します。卵の殻を破ることがないように注意してください。小さな小窓を作るために一つずつ削除します。慎重に卵黄膜に触れずに鉗子を用いて内膜を除去します。 </li></ol> 2。注射とエレクトロ<ol><li>脳や心臓の損傷を防ぐために、血管が目に入ると、くちばしに向かって指しているのメインバンドルの横針コントラ配置します。 </li><li>針の突然の穏やかなプッシュによって卵黄膜、強膜、網膜と硝子体を貫く。針が目のもう一方の端を突き抜けている場合は、任意の主要な血液の静脈を傷つけない限りは大丈夫でなければなりません。 </li><li>徐々に開口部の端に針を引き戻すと、眼球の外側の壁にはほぼ接線に配置します。 </li><li>差し込み、トン強膜と網膜の間にサブ網膜スペースに針。 </li><li>あなたが眼球の側を充填し、膨らみを作成することによって、網膜の内側を押して緑色の溶液を可視化できるようになるまでのDNAを注入します。 </li><li>あなたの針の打込みが正しくない場合、あなたは眼球の中間を埋める硝子体内部に拡散したDNA溶液が表示されます。あなたがあまりにも多くの網膜が損傷した場合にも、その後、DNA溶液は、眼球から出てくるが表示されます。 </li><li>徐々に針を除去し、直ちにPBSに浸漬した後に卵の内部で並列に電極を配置します。注入された目が電極間に配置されるようにように、羊水に沈めるに電極を押し下げます。それらを配置しながら、電極とすべての主要な血管や心臓には触れないでください。そのDNAはサブ網膜スペースから正極に向かって網膜に輸送できるように、負極は、注入側でなければなりません。エレクトロ950ミリ秒間隔で50ミリ秒間15Vの5パルスで網膜をporate。 </li><li>慎重に電極を除去し、透明なセロハンテープの部分と卵の窓をシールします。 </li><li>ラベルとインキュベーターに戻し入れる前に注入された卵日付を記入してください。一般的に、それは全体のエレクトロポレーションプロセスを完了するために約3〜5分かかります。 </li><li> GFPの発現は、エレクトロポレーション後8時間には早くも見ることができます。胚は収穫前に、必要な段階に達するまで、ただし、待つことがあります。 </li></ol> 3。代表的な結果私たちの研究では、網膜細胞の開発に関与する遺伝子発現の調節を研究するために様々なプラスミドコンストラクトを使用しています。このビデオでpCAG-GFP（トランスフェクションコントロール）が成功した注入とエレクトロポレーションに従うように使用されていました。しかし、レポーター遺伝子（GFP、RFPなど）を持つ任意のプラスミドコンストラクトを使用することができます。 GFPの発現はELE 8時間後には早くも見ることができるにもかかわらずctroporation、我々は、通常、6日目（E6）と以降の卵を収穫を開始します。エレクトロポレーションの網膜は、胚の切り出しと埋め込みとセクショニングの前に蛍光解剖顕微鏡下で分析した。一般的に、レポーター遺伝子の発現が成功したエレクトロポレーション（図1）後、少なくとも網膜の四半期で見ることができます。トランスフェクトされた網膜組織はさらに、細胞の形態を明確に可視化するために切片を介して分析した。細胞型特異的なマーカーを用いた免疫組織化学（Brn3a、Pax6の等）を細胞特異的GFP発現の許容特性（図2）。 <img src="/files/ftp_upload/3792/3792fig1.jpg" align="Figure 1" /> 図1。レポータープラスミド結果正のGFP発現の成功したエレクトロポレーション。ニワトリ胚網膜を注入し、エレクトロポレーション胚の4日目に、胚6日目に収穫しました。網膜の少なくとも25％が正常にトランスフェクトした（=トップビュー、B=ボトムビュー）。スケールバー= 1ミリメートル。 <img src="/files/ftp_upload/3792/3792fig2.jpg" align="Figure 2" /> 図2。免疫組織化学を用いた網膜細胞を発現しているGFPの特性。GFP E7段階で網膜組織を表現するには、固定と切片。これらのセクションは、さまざまな細胞特異的マーカーで染色した。 Brn3aはPax6のは水平、アマクリンおよび神経節細胞（B）を決定するために使用している間に神経節細胞（）を決定するために使用されていました。スケールバー=50μmである。

<ol>
	<li>Doh, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+retinal+cell+development+in+chick+embryo+by+immunohistochemistry+and+in+ovo+electroporation+techniques.">Analysis of retinal cell development in chick embryo by immunohistochemistry and in ovo electroporation techniques.</a> BMC. Dev. Biol. 10, (2010).</li><li>Blank, M. C., Chizhikov, V., Millen, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Ovo+Electroporations+of+HH+Stage+10+Chicken+Embryos.">In Ovo Electroporations of HH Stage 10 Chicken Embryos.</a> J. Vis. Exp. (9), e408-e408 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</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.+1951.">A series of normal stages in the development of the chick embryo. 1951.</a> Dev. Dyn.. 195, 231-272 (1992).</li><li>Hamburger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stage+series+of+the+chick+embryo.">The stage series of the chick embryo.</a> Dev. Dyn. 195, 273-275 (1992).</li></ol>

動物実験のすべては、ラトガーズ大学の動物実験および施設委員会によって承認されました。

網膜の注入を対象とE4段階での胚の網膜でのOVOエレクトロポレーションでは特に単一細胞レベルでのすべての6つの主要な網膜細胞型を視覚化する能力が得網膜前駆細胞をターゲットにすることができます。OVOエレクトロポレーションでターゲティングHH10（〜E1.5）で眼胞は眼を形成するために開発した細胞をトランスフェクトすることができます。しかし、これら?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>受精した病原体フリー（SPF）白色レグホン鶏の卵</td><td>サンライズファーム（キャッツキル、ニューヨーク州） </td><td></td><td></td></tr><tr><td>鶏の卵のインキュベーター</td><td> GQF製造、サバンナ、ジョージア州</td><td>モデル-1550 </td><td> 60％の湿度と37.5℃に設定</td></tr><tr><td>ガラスキャピラリーチューブ</td><td>世界精密計測器株式会社</td><td> TW150F-4 </td><td></td></tr><tr><td> 0.1mlの注射器</td><td>ハミルトン（株）リノ、ネバダ州</td><td>気密1710 </td><td>あるいは、1ミリリットル注射器を使用することもでき</td></tr><tr><td> Masterflexシリコーン（過酸化水素）チューブ</td><td>コー​​ル·パーマー</td><td> HV-96400から13 </td&gt; <td>注射器にガラス針を取り付けるための小片（1 cm）をカット</td></tr><tr><td>ピンセット</td><td>デュモン</td><td>単3 </td><td></td></tr><tr><td>マニピュレーター</td><td>世界精密計測器株式会社</td><td> M3301-M3 </td><td></td></tr><tr><td>プラスミドDNA </td><td></td><td></td><td> pCAG-GFPは、博士コニーCepkoから借用したもの</td></tr><tr><td>ファストグリーンFCF </td><td> Sigma-Aldrich社</td><td> F7252 </td><td> PBSで0.025パーセントに希釈する</td></tr><tr><td>パルス発生器</td><td>ハーバード装置、MA </td><td> BTX ECM 830 </td><td>方形波発生器</td></tr><tr><td>電極</td><td>ハーバード装置、MA </td><td> BTXモデル514 </td><td>私たちの電極は、3〜5ミリメ​​ートル間隔をあけた</td></tr><tr><td>モノクロデジタルカメラ</td><td>ツァイス、ドイツ</td><td> Axiocam MRM </td><td></td></tr><tr><td>蛍光解剖顕微鏡</td><td>ライカ、ドイツ</td><td>ライカMZ16FA </td><td></td></tr><tr><td>直立蛍光顕微鏡</td><td>ツァイス、ドイツ</td><td>ツァイスアクシオイメージャA1 </td><td></td></tr></tbody></table>

in ovo electroporation in embryonic chick retina

ニワトリ胚網膜は高等脊椎動物の網膜の開発を研究する優れたツールです。ので、大きいサイズと、外部の開発に、それは、組換えDNA / RNA技術を用いたニワトリ胚網膜を操作するために比較的非常に簡単です。胚の網膜へのDNA / RNAの構造のエレクトロポレーションは、網膜の開発時に網膜幹細胞/前駆細胞の遺伝子調節を研究するための大きな利点を持っています。このようなレポーター遺伝子アッセイ、過剰発現、遺伝子等（shRNA）をノックダウンする遺伝子としてアッセイの異なるタイプのエレクトロポレーション技術を用いて行うことができます。このビデオは、対象となる網膜の注入およびOVOエレクトロポレーションのハンバーガーで、ニワトリ胚の網膜に、胚4日目（E4）であるハミルトンのステージ22から23を示しています。マーカーとして緑色蛍光タンパク質（GFP）を発現するプラスミドDNAを直接に配信されることにより、ここでは、OVOエレクトロポレーション技術で 、迅速かつ便利を表示ニワトリ胚網膜下腔、網膜幹細胞/前駆細胞によるDNAの取り込みを容易にするために、電気パルスが続く。 E4で網膜注入とエレクトロポレーションの新しいメソッドは、E1.5 2で注入し、エレクトロポレーションの従来の方法では困難であった後半生まれのニューロン1、を含む、すべての網膜細胞の種類、可視化することができます。

Watch this Scientific Journal Video about In Ovo Electroporation in Embryonic Chick Retina at JoVE.com

In Ovo Electroporation in Embryonic Chick Retina

このビデオの全体的な目標は、対象と網膜の注入を実行する方法を示すことであると<emOVOで&gt;</em&gt;ハンバーガーで、ニワトリ胚の網膜へのDNA / RNAの構造のエレクトロポレーションおよび胚4日目（E4）であるハミルトンのステージ22-23日。この手法は、遺伝子発現、遺伝子調節、ニワトリ網膜の開発の形態変化を研究する非常に便利です。

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 ...

in-ovo-electroporation-in-embryonic-chick-retina

Research

JoVE Journal

Biology

In Ovo Electroporation in Embryonic Chick Retina

14.3K ビュー.  University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School. このビデオの全体的な目標は、対象と網膜の注入を実行する方法を示すことであるとハンバーガーで、ニワトリ胚の網膜へのDNA / RNAの構造のエレクトロポレーションおよび胚4日目（E4）であるハミルトンのステージ22-23日。この手法は、遺伝子発現、遺伝子調節、ニワトリ網膜の開発の形態変化を研究する非常に便利です。

ビデオ: In Ovo Electroporation in Embryonic Chick Retina

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

HHステージ10ニワトリ胚のOVOエレクトロポレーションで

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

生物学

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

鶏胚における神経誘導のためのアッセイ

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

チック EX卵文化と EX卵 CAMアッセイ：それが実際にどのように動く

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

活用チック神経管の神経細胞の遺伝的に定義されたグループの軸索経路の解読卵のエレクトロポレーション

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

ヒト胚性幹細胞のコロニーの成長のビデオバイオインフォマティクス解析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

鶏胚における単一細胞のトランスフェクション

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

初期のニワトリ胚の形態形成の組織の変形を追跡

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...