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1 例OVO文化<ol><li>新鮮な受精卵は、インキュベーションのために60％の湿度で37.5で強制ドラフトインキュベーター（BSS160、エレット、ドイツ）°Cでの長い側に配置されます。より長い一週間に12℃で保存した卵は使用すべきではありません。 </li><li> 2.5日（ステージHH17程度）のインキュベーション後、卵を取り出し、クラックの方向性を示すために、鉛筆で上部にラベルを付けます。 </li><li>クラッキングの前に、きれいな大型のペトリ皿（;グライナーバイオワン社、ドイツ145ミリメートルの直径）に約20 mlの滅菌蒸留水を注ぐ。 </li><li>大規模なペトリ皿に、別の小さな滅菌シャーレ（グライナーバイオワン社94ミリメートルの直径）を配置します。 </li><li>鋭い金属エッジに対して底部に卵を割る、慎重に卵を開いて、小さなペトリ皿にそれぞれの卵の内容全体を転送します。 </li><li>蓋付きの大きなペトリ皿をカバーし、別のインキュベーターに入れて（BINDER社、Tuttlinge、ドイツ）さらに文化の37.5℃で約60％の湿度である。日、所望の数のインキュベーションの後、胚はエレクトロポレーションに使用することができます。 </li></ol> 2。 元OVOエレクトロポレーションの準備<ol><li> 1.0 mm径のガラス管を使用して（TW100F-4、世界の精密機器）微小電極プーラー（ワールド·精密機器、ベルリン、ドイツPUL-100マイクロピペットプラー）でガラスキャピラリーを引き出し、適切にガラスキャピラリーの先端を破る直径。 </li><li>標的組織および胚の大きさに応じて、エレクトロポレーションのために適切なパラメータを（異なる組織および胚の大きさのために、すなわち、異なる電圧）を設定し、エレクトロに電極を接続します（NEPAジーン、千葉県、日本CUY21·エディット）。 </li><li>とマーカー遺伝子、ターゲット遺伝子をコードするpCAGGSベクターのプラスミド、例えばcadherin7（2.0μg/μLの濃度のpCAGGS-Cad7 Cad7）を含むプラスミド溶液を調製例えば、緑色蛍光タンパク質（GFP、0.25μg/μLの濃度のpCAGGS-GFP）。その後、緑の色でプラスミド溶液にラベルを付けるために最終濃度0.1％（Sigma）でファストグリーンを追加します。 </li><li>口のピペットを用いてプラスミド溶液とガラスキャピラリーをロードします。 </li></ol> 3。 元OVOエレクトロポレーションによるニワトリ視蓋への遺伝子導入<ol><li> 元OVO培養後、例えば、インキュベーション日（E）6によって、ニワトリ胚は 、ex OVOエレクトロポレーションに使用されています。 </li><li>慎重に微細な鉗子で視蓋の上に卵黄と羊膜を引き裂き、蓋の表面に滅菌した0.9％塩化ナトリウム溶液3滴を追加します。 </li><li>口のピペットを用いてガラスキャピラリーによる蓋の空洞にプラスミド溶液を注入します。 </li><li>タングステンニードルカソードと蓋の横にある長方形の板のアノード（CUY 661-3x7、NEPAジーン）で電極を配置し、すぐに蓋にエレクトロによって生成された電気パルス（6パルス、25 V、60 msパルスの長さ、それぞれのケースでは100ミリ秒間隔）を適用します。 </li><li>蓋付きの大きなペトリ皿をカバーし、インキュベーターに戻すことができます。適切な日数（例えば、2または3日エレクトロポレーション後）のインキュベーションの後、蓋を免疫染色（ 図1）と生物学的検出のために収集され、固定されています。 </li></ol> 4。代表的な結果 元OVOエレクトロポレーションによるCad7とGFPの外因性タンパク質の過剰発現が成功した例として、図1に示されています。プラスミドGFPと一緒にCad7プラスミドときには、胚を採取し、固定した二、三日後、エレクトロポレーション、E6で蓋にエレクトロポレートした。 E8では、GFPタンパク質が強く全体のマウント画像の蓋（ 図1A-C）で表されます。また、E9で蓋のセクションで、GFPタンパク質（ 図1Dの緑）とCad7タンパク質（ 図1Eの赤）EX OVOエレクトロポレーションは 、in vivo での古いニワトリ胚への遺伝子導入のために成功した方法であることを示唆している（ 図1Fの黄色）共発現である。 <img src="/files/ftp_upload/4078/4078fig1.jpg" alt="図1"/> E8でwholemount画像（AC）に示すように、 図1。プラスミドのエンコーディングGFPとCad7が二、三日の元のOVOエレクトロポレーションした後、E6で鶏の蓋にコトランスフェクトされた場合、GFPタンパク質（緑）が強く発現されています。 E9で蓋のセクションでは、GFPタンパク質（Dの緑）とCad7タンパク質（Eの赤）（Fの黄色）共発現されています。 BF、明視野像。スケールバー：AC用では200μm、DFのDで100μmである。

<ol>
	<li>Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+gene+expression+in+chick+embryos+by+microelectroporation.">Efficient targeting of gene expression in chick embryos by microelectroporation.</a> Dev. Growth Differ. 41, 335-344 (1999).</li><li>Luo, J., Ju, M. J., Lin, J., Yan, X., Markus, A., Mix, E., Rolfs, A., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-20+expression+by+motor+neurons+is+regulated+by+Sonic+hedgehog+during+spinal+cord+development.">Cadherin-20 expression by motor neurons is regulated by Sonic hedgehog during spinal cord development.</a> NeuroReport. 20, 365-370 (2009).</li><li>Luo, J., Ju, M. J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regionalized+cadherin-7+expression+by+radial+glia+is+regulated+by+Shh+and+Pax7+during+chicken+spinal+cord+development.">Regionalized cadherin-7 expression by radial glia is regulated by Shh and Pax7 during chicken spinal cord development.</a> Neuroscience. 142, 1133-1143 (2006).</li><li>Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., 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=Gain-+and+loss-of-function+in+chick+embryos+by+electroporation.">Gain- and loss-of-function in chick embryos by electroporation.</a> Mech. Dev. 121, 1137-1143 (2004).</li><li>Dai, F., Yusuf, F., Farjah, G. H., Brand-Saberi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi-induced+targeted+silencing+of+developmental+control+genes+during+chicken+embryogenesis.">RNAi-induced targeted silencing of developmental control genes during chicken embryogenesis.</a> Dev. Biol. 258, 80-90 (2005).</li><li>Rao, M., Baraban, J. H., Rajaii, F., Sockanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+comparative+study+of+RNAi+methodologies+by+in+ovo+electroporation+in+the+chick+embryo.">In vivo comparative study of RNAi methodologies by in ovo electroporation in the chick embryo.</a> Dev. Dyn. 231, 592-600 (2004).</li><li>Kos, R., Tucker, R. P., Hall, R., Duong, T. D., Erickson, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+introducing+morpholinos+into+the+chicken+embryo.">Methods for introducing morpholinos into the chicken embryo.</a> Dev. Dyn. 226, 470-477 (2003).</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> J. Morphol. 88, 49-92 (1951).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 10, 391-394 (1974).</li><li>Luo, J., Treubert-Zimmermann, U., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+guide+migrating+Purkinje+cells+to+specific+parasagittal+domains+during+cerebellar+development.">Cadherins guide migrating Purkinje cells to specific parasagittal domains during cerebellar development.</a> Mol. Cell. Neurosci. 25, 138-152 (2004).</li><li>Luo, J., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+electroporation+for+gene+transfer+into+older+chicken+embryos.">Ex ovo electroporation for gene transfer into older chicken embryos.</a> Dev. Dyn. 233, 1470-1477 (2005).</li><li>Treubert-Zimmermann, U., Heyers, D., Redies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+axons+to+specific+fiber+tracts+in+vivo+by+altering+cadherin+expression.">Targeting axons to specific fiber tracts in vivo by altering cadherin expression.</a> J. Neurosci. 22, 7617-7626 (2002).</li></ol>

利害の衝突が宣言されません。

このプロトコルは、 元のOVOエレクトロポレーションによって、古いニワトリ胚への遺伝子導入（例えば、E6で視蓋に）のためのガイドを提供します。このメソッドは古い鶏胚にエレクトロポレーションの使用を拡張し、簡単に後の段階でin vivoでの遺伝子機能を研究するための多くの研究室に適用することができます。 E4から少なくともE7の胚は正常にこのメソッドによってエレ?...

<table border="1"><tbody><tr><td> 名 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>エレクトロ</td><td> NEPAジーン、日本</td><td> CUY21·編集</td></tr><tr><td>電極</td><td> NEPAジーン、日本</td><td> CUY661-3x7 </td></tr><tr><td>卵インキュベーター</td><td>エレット、ドイツ</td><td> BSS160 </td></tr><tr><td>胚インキュベーター</td><td>バインダーGmbH、ドイツ</td><td></td></tr><tr><td>蛍光顕微鏡</td><td>キーエンス、Deutschland GmbHは</td><td> BZ-8000 </td></tr><tr><td>ペトリ皿</td><td>グライナーバイオワン社、ドイツ</td><td></td></tr><tr><td>微小電極プラー</td><td>世界精密機器、ドイツ</td><td> PUL-100 </td></tr></tbody></table>

gene transfer into older chicken embryos by ex ovo electroporation

ニワトリ胚は、胚発生時の遺伝子の機能と調節を研究するための優れたモデルシステムを提供します。OVOエレクトロポレーションで過剰発現する外来遺伝子に強力なメソッドまたはニワトリ胚1のin vivoでダウンレギュレートする内因性遺伝子である。このようなDNAプラスミドをコードする遺伝子2-4、低分子干渉RNA（siRNA）のプラスミド5、小さな合成RNAオリゴ6、モルホリノ、アンチセンスオリゴヌクレオチド7と異なる構造を簡単にエレクトロポレーションによってニワトリ胚にトランスフェクトすることができます。しかし、OVOエレクトロポレーションでのアプリケーションは、初期のインキュベーション段階（ステージHH20未満-ハンブルクとハミルトンによる）で胚に限定されている8およびそれ以降の段階で胚への応用のためのいくつかの欠点は、（ステージHH22歳以上あります-約3.5開発の日）。たとえば、後の段階では卵黄膜は有珠山である味方SHALL膜に付着し、シェルウィンドウを開くには、胚の死、その結果、血管の破裂を引き起こす。古い胚は、それが胚にアクセスして操作することは困難である卵黄と尿膜の血管によって覆われている、古い胚が移動精力的に、シェルでは比較的小さな窓から配向性を制御することは困難である。 このプロトコルでは、後期段階（ステージHH22歳以上）でニワトリ胚への遺伝子導入のために元のOVOエレクトロポレーション法を示しています。 元OVOエレクトロポレーションのために、胚はペトリ皿9に培養されており、卵黄や尿管が広く普及しています。これらの条件下では、古いニワトリ胚は、簡単にアクセスおよび操作されます。したがって、このメソッドは、以前のニワトリ胚に適用されるOVOエレクトロポレーションでの欠点を克服しています。この方法を使用して、プラスミドを容易に異なる部分にトランスフェクトすることができます古いニワトリ胚10-12。

Watch this Scientific Journal Video about Gene Transfer into Older Chicken Embryos by ex ovo Electroporation at JoVE.com

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

後のインキュベーション段階（ハンバーガーとハミルトンステージ（HH）22歳以上）でニワトリ胚への遺伝子導入方法が記載されています。このメソッドは次の欠点を克服する<emOVOで&gt;</em&gt;エレクトロポレーションは、古いニワトリ胚に適用され、古い発達の段階で遺伝子の機能と調節を研究するための便利なテクニックです。

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 ...

gene-transfer-into-older-chicken-embryos-by-ex-ovo-electroporation

Research

JoVE Journal

Biology

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

14.5K ビュー.  School of Medicine University of Rostock. 後のインキュベーション段階（ハンバーガーとハミルトンステージ（HH）22歳以上）でニワトリ胚への遺伝子導入方法が記載されています。このメソッドは次の欠点を克服するエレクトロポレーションは、古いニワトリ胚に適用され、古い発達の段階で遺伝子の機能と調節を研究するための便利なテクニックです。

ビデオ: Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

マウス卵母細胞への核移転

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

生物学

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

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

E15マウス胚の子宮内での脳室内注入とエレクトロポレーションで

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

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

アーリーステージのニワトリ胚に成長因子コートビーズの配置

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

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 ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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 ...

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

ニワトリ胚へのヒト幹細胞の異種移植

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

アン EX - OVO</emイメージングとマイクロサーアプリケーションに適して>ニワトリ胚培養システム

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...