我々は彼らのサポートとこの原稿について有益なコメントをBarresiラボのメンバーに感謝したいと思います。私たちは、スミスカレッジゼブラフィッシュのコロニーを維持する上で彼らの助けのための彼の一定の技術支援だけでなく、動物のケアスタッフのためのアレクサンダーワークマンに感謝。我々はまた、このプロトコルのfilminためのいくつかの顕微鏡の機器を貸与するためのマイクのHallacy、ルディRottenfusser、そしてカールツァイスマイクロイメージングに感謝。この作品は、NSFの資金による研究助成金、0615594によってサポートされていました。

<ol>
	<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+for+the+laboratory+use+of+zebrafish+(Danio.">A guide for the laboratory use of zebrafish (Danio.</a> The Zebrafish Book. , (2007).</li><li>N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+A+Practical+Approach.">Zebrafish A Practical Approach.</a> , (2002).</li><li>Graeden, E., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> J Vis Exp. (26), (2009).</li><li>Gilmour, D. T., Jessen, J. R., Lin, S., N&#252;sslein-Volhard, C., Dahm, R. e. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish,+a+practical+approach.">Zebrafish, a practical approach.</a> 1, 121-121 (2002).</li><li>Woo, K., Shih, J., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+maps+of+the+zebrafish+embryo.">Fate maps of the zebrafish embryo.</a> Curr Opin Genet Dev. 5 (4), 439-439 (1995).</li><li>Barresi, M. J., Hutson, L. D., Chien, C. B., Karlstrom, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+regulated+Slit+expression+determines+commissure+and+glial+cell+position+in+the+zebrafish+forebrain.">Hedgehog regulated Slit expression determines commissure and glial cell position in the zebrafish forebrain.</a> Development. 132 (16), 3643-3643 (2005).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75 (5), 551-551 (1997).</li><li>Halloran, M. C., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+gene+expression+in+specific+cells+of+transgenic+zebrafish.">Laser-induced gene expression in specific cells of transgenic zebrafish.</a> Development. 127 (9), 1953-1953 (2000).</li><li>Hardy, M. E., Ross, L. V., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+gene+misexpression+in+zebrafish+embryos+induced+by+local+heat+shock+using+a+modified+soldering+iron.">Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.</a> Dev Dyn. 236 (11), 3071-3071 (2007).</li></ol>

The authors have nothing to disclose.

キメラ胚を生成すると、いくつかのモデル系、すなわちミバエ（D.melanogaster）、ワーム（ 虫）、ゼブラフィッシュ（D.rerio）とマウス（M.musculus）内の多くの研究課題に対応する強力なツールです。我々は、ゼブラフィッシュのモデルシステムを一意に比較的容易、迅速かつ汎用性の高い方法でキメラを生成するために適していると主張している。ゼブラフィッシュはマウスモデルと比較して、それはかなり利用しやすく、母親の外側に開発脊椎動物です。ゼブラフィッシュはまた、細胞移植などの発生学的操作を簡素化するハエやワーム、よりもはるかに大きいです。さらに、トランスジェニック技術と細胞移植の手順を結合する機能は、ローカライズされた組織特異的に操作できるようにする遺伝子の機能で可能な実験の大規模な配列が可能になります。例えば、GFP +またはローダミン標識された細胞の小さなクローンはしばしばホモ接合トランスジェニックドナーに失われるか、または一般的な抗体を用いた差動細胞内標識による可視化することは不可能であるしている完全な細胞の形態の特性評価を可能にします。さらに、トランスジェニックタグ付きまたは蛍光充填されたセルを使用することによって、我々は時間をかけてライブゼブラフィッシュ胚にドナー細胞を追跡することができます。個々の細胞のタイムラプスイメージングは​​生物全体の文脈における細胞挙動の小説の分析を提供します。 私たちの研究室はまた、熱ショックを組み合わせて使用​​、特定の軸索ガイダンスの手がかりは、培養温度の上昇（データは表示されません）以下のドナーの細胞でmisexpressedすることができるように細胞移植によるトランスジェニック系統を誘導性。この手法は、局所的に私たちは特定のタンパク質が開発にどのように影響するかを見ることができる時間的空間的固有の方法で目的のタンパク質をmisexpressすることは非常に強力で直接的なアプローチです。私たちのケースでは、この手法は、正中線5の交連とグリア細胞の位置におけるスリットロボの指導の手がかりの機能の背後にある我々の知識を拡張することができます。 このような焦点UVアンケージングとローカライズされたヒートショックのツール（レーザーやはんだごてなど）などのローカルmisexpressingの遺伝子に他のアプローチは、遺伝子や細胞7-9の小さなクラスタ内のマーカーの発現を操作するための代替方法を提供するかが、細胞移植は確立細胞移植より制御された実験的なアプローチを作る紫外線、レーザーまたは熱によって誘発されるあらゆる外傷のない分析の最終的な環境。結論として、我々は神経生物学研究の重要な一部となる細胞移植の手順を発見した。異なる特性を持つ細胞のクローンを生成すると、細胞の挙動、遺伝子機能、および細胞の自律性の基本的な問題に対処するための発生生物学者のための重要なツールです。

<table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> の名前 </td><td> タイプ </td><td> 会社 </td><td> カタログ番号 </td><td> 具体的な </td></tr><tr><td>ペトリ皿</td><td>ツール</td><td> Fisherbrand </td><td> 08-757-13 </td><td> 100 × 15ミリメートル</td></tr><tr><td>グラスの底培養プレート</td><td>ツール</td><td>マテック</td><td> P35G - 1.5 - 2.0 - C </td><td> 35 × 15ミリメートル、なし1.5、コーティングされていない、ガンマ線照射</td></tr><tr><td>ワイドボアガラスパスツールピペット</td><td>ツール</td><td> Fisherbrand </td><td> 63A - 53 - WT </td><td></td></tr><tr><td>トラフモールド用ガラスフィラメント毛細血管（糸なし）と注射針（フィラメント付き） </td><td>ツール</td><td>世界の精密機器</td><td> 1B150F - 4 </td><td> 4インチ（100mm）を、1.5 / 0.84外径/内径（mm） </td></tr><tr><td>移植キャピラリー（TransferTip） </td><td>ツール</td><td>エッペンドルフ</td><td> 83000122から8 </td><td>タイプIV、滅菌は、Int。 15μmの、20度曲げ</td></tr><tr><td>移植の鋳型</td><td>ツール</td><td>適応科学ツール</td><td> PT - 1 </td><td>個々の胚を保持するために150三角divots </td></tr><tr><td>アガロースは、タイプI </td><td>試薬</td><td>シグマアルドリッチ</td><td> A6013 - 250G </td><td> 1.5％アガロースは、EMで行わ</td></tr><tr><td> （抗生物質）胚のミディアム</td><td>試薬</td><td></td><td></td><td>ウェスター、2007を参照してください。 </td></tr><tr><td>リン酸塩緩衝液</td><td>試薬</td><td></td><td></td><td>ウェスター、2007を参照してください。 </td></tr><tr><td>ウォッチマン鉗子</td><td>ツール</td><td>ファイン科学ツール</td><td></td><td> 100 +ミリメートルチップ（鈍い）、50ミリメートルのヒント</td></tr><tr><td>ローダミンBデキストラン</td><td>リネージュトレーサー色素</td><td>分子プローブ、インビトロジェン</td><td> D1824 </td><td> MW万ダ、リジン - 修正可能、赤（590nm）、緑色（570nm）の光で励起された蛍光色素</td></tr><tr><td>解剖顕微鏡</td><td>顕微鏡</td><td>オリンポス</td><td> SZX7 </td><td></td></tr><tr><td>蛍光実体顕微鏡</td><td>顕微鏡</td><td>ツァイス</td><td> Lumar </td><td>完全に自動化されたジョイスティック制御（Sycop） </td></tr><tr><td>移植装置</td><td>ツール</td><td>エッペンドルフ（株） </td><td> AirTram </td><td></td></tr><tr><td>自動化されたマイクロ</td><td>ツール</td><td>エッペンドルフ（株） </td><td> TransferMan NK2 </td><td>ジョイスティック制御</td></tr><tr><td>マニピュレーター</td><td>ツール</td><td>アプライドサイエンティフィックインスツルメンツ株式会社</td><td> 00-42-101-0000 </td><td>右MM33 </td></tr><tr><td>バックプレッシャーユニットとマイクロインジェクター</td><td>ツール</td><td>アプライドサイエンティフィックインスツルメンツ株式会社</td><td> MPPI - 2 BPU </td><td></td></tr><tr><td> Flamming /ブラウンマイクロピペットプラー</td><td>ツール</td><td>サター器械公司</td><td>モデルP97 </td><td>プログラム：ヒート550; 速度200; 時間/ディレイ200; 力120を引き出します。 </td></tr></tbody></table>

tissue targeted embryonic chimeras: zebrafish gastrula cell transplantation

細胞が新たな環境に置かれているか、より大きな文脈の中の細胞の小さなグループが変更されているときに時発生生物学の分野で一定の基本的な質問にだけ答えることができます。一つのセルが相互作用し、ユニークな環境の中でどのように動作するかを見ることは細胞機能を特徴づけるために不可欠です。どのように細胞を取り巻く特定のタンパク質の影響のローカライズmisexpressionを決定することはタンパク質は発生過程の様々で果たす役割についての洞察力に富んだ情報を提供します。私たちの研究室では独自に遺伝子型または表現型のキメラを生成するために古典的な移植技術と遺伝的アプローチを組み合わせることがゼブラフィッシュのモデルシステムを使用しています。我々は、ゼブラフィッシュにおける前脳の交​​連を形成する際にニューロングリア細胞の相互作用を研究する。このビデオは私たちの研究室は、アストログリア間脳の集団と彼らは正中線を横切るように軸索を投射に影響を与える特定の指導の手がかりの役割の役割を調査するための方法を説明します。その透明度のゼブラフィッシュ胚に起因する異所性細胞の配置またはローカライズされた遺伝子のmisexpressionのこのタイプの理想的なモデルです。追跡移植された細胞は生体染色色素や蛍光タンパク質を発現するトランスジェニック魚のラインを使用して行うことができます。我々は、移植のための重要な色素トレーサーとドナー胚を準備する方法ここで示すだけでなく、1つ原腸胚から細胞を抽出して移植する方法を別の胚を上演。我々は、ゼブラフィッシュ胚の前脳内に異所性GFP +トランスジェニック細胞を示すデータを提示し、交連を前脳に関して、これらの細胞の位置を特徴づける。さらに、我々はGFP +トランスジェニック宿主胚に移植アレクサ594ラベルされた細胞のレーザー走査型共焦点タイムラプス顕微鏡を示しています。これらのデータは、原腸胚段階移植は、発生生物学のさまざまな質問に対処するために異所性細胞の標的位置決めを可能にするという証拠を提供する。

Watch this Scientific Journal Video about Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation at JoVE.com

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

ゼブラフィッシュ細胞移植は、組織特異的キメラを生成するために遺伝学と発生学の組み合わせを可能にします。このビデオでは、原腸胚の研究室では前脳の交連形成時のアストログリア集団と特定の指導の手がかりの役割を調査することを許可している細胞移植を段階を示しています。

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

tissue-targeted-embryonic-chimeras-zebrafish-gastrula-cell

Research

JoVE Journal

Biology

19.2K ビュー.  Smith College. ゼブラフィッシュ細胞移植は、組織特異的キメラを生成するために遺伝学と発生学の組み合わせを可能にします。このビデオでは、原腸胚の研究室では前脳の交連形成時のアストログリア集団と特定の指導の手がかりの役割を調査することを許可している細胞移植を段階を示しています。

ビデオ: Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

アダルトゼブラフィッシュの全腎骨髄の移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

生物学

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

アクチン共沈降アッセイ、F -アクチンへの結合タンパク質の解析のための

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植によるキメラゼブラフィッシュ胚の生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

ゼブラフィッシュ胚眼の組織の顕微解剖

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

キメラの生成のためのメダカ胚および細胞移植のDechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

トランスジェニックゼブラフィッシュの多色タイムラプスイメージング：標的神経細胞のアブレーションによって活性化を可視化網膜幹細胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

から初代細胞培養ショウジョウバエ原腸胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接アダルトゼブラフィッシュの腎臓への細胞の移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis1. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample2, 3. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. 
Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease4. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression5. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies 6-8. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency9, 10. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish8 and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency.
Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

同系ゼブラフィッシュにおける限界希釈細胞移植を用いた腫瘍増殖細胞の頻度を定量化

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

ディテールに秘められたゼブラフィッシュ胚心臓の免疫染色

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...