Name: use of the teton system to study molecular mechanisms of zebrafish regeneration
Uploaded: 2015-06-25T13:00:00.000+00:00
Duration: 10 min 22 s
Description: Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

著者は、技術支援のためのクリスタハーゼ、ドリス·ウェーバーとブリジットKorteの感謝します。ワイディンガーラボでの仕事はドイツ学術協会WE 4223 / 3-1の補助金によってサポートされ、4223 / 4-1とドイツゲゼルシャフトエリーゼKardiologieオスカー·ラップ·Stipendiumを経由し、クラウス·ゲオルクウント·Sigrid-によりWE Hengstberger-Forschungsstipendium。

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+2A+peptides+allow+expression+of+multiple+proteins+from+a+single+ORF+in+transgenic+zebrafish+embryos.">Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.</a> Genesis. 45 (10), 625-629 (2007).</li><li>Suster, M. 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R., Thummel, R. <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+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. (61), (2012).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610 (2007).</li><li>Thermes, V., 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=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Suster, M. L., Kikuta, H., Urasaki, A., Asakawa, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Mol Biol. , 56141-56163 (2009).</li><li>Grabher, C., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meganuclease+and+transposon+mediated+transgenesis+in+medaka.">Meganuclease and transposon mediated transgenesis in medaka.</a> Genome Biol. 8, (2007).</li></ol>

著者らは、開示することは何もありません。

大人のゼブラフィッシュは、正常に多くの内臓や付属を再生する驚くべき能力を持っています。関与する分子·細胞メカニズムの完全な理解は、遺伝子の機能とシグナル伝達経路の組織特異的な分析が必要です。この方に、ティトンシステムは、胚および成体ゼブラフィッシュにおける時空間的制御遺伝子発現のための効率的なツールを提供します。この原稿に記載されテトン·システムの構...

ゼブラフィッシュは、開発、生理学、疾病、および再生の多くの側面を研究するための十分に確立された脊椎動物のモデル生物です。後胚生物学的プロセスのモデルとしてゼブラフィッシュの成長採用により、遺伝子発現またはシグナル伝達経路の誘導性、組織特異的な操作のための実験的なツールは、ますます重要になってきました。特に、成人のゼブラフィッシュにおける臓器や付属物の再生への研究は、これらの再生プロセスの間、シグナル伝達経路の時空間的な要件の解剖のためのツールの不足に苦しんでいます。 のCre-lox系、トランスポゾン媒介体形質転換を用いて、熱ショック誘導性導入遺伝子のモザイク発現、及びティシステム1：現在、三つの異なるシステムが成体ゼブラフィッシュの器官の再生の条件、組織特異的遺伝子発現を達成するために使用されています-3。ティトンはテトラサイクリン続きの変種を指します発現は、抗生物質テトラサイクリンまたはその誘導体、 例えば、ドキシサイクリンの存在下で活性化されたロール転写活性化システム。それまで成魚に使用されているようのCre-lox系は、その発現が空間的に組織特異的調節エレメントによって制限されているタモキシフェン制御Creリコンビナーゼ（CreERT2）に依存しています。 STOPカセットのCreドリブン除去は、全ての細胞型1でアクティブであるべきであるプロモーターによって駆動される目的の遺伝子の発現を促進します。モザイク状に発現体細胞導入遺伝子のトランスポゾン媒介性の作成は、個々の細胞系統における誘導性導入遺伝子発現のためのシステムを提供します。一般的にのみ再生臓器2の離散的な細胞系統に導入遺伝子を有するキメラ個体においてヒートショックプロモーター結果の転写制御下に目的の遺伝子を運ぶのTol2トランスポゾンとゼブラフィッシュ胚の注入。両方のシステムは、条件付き組織-SPEを可能にしながら、cific遺伝子発現、のCre-lox系は可逆的ではない、とトランスポゾンクローンの標識を使用しての戦略は、その確率的性質に苦しんでいます。このように、私たちと他の人は最近、時間的に容易に、ゼブラフィッシュで使用するためのトランスジェニックテトン·システム、および添加調整可能と可逆3-5にある空間的に制御された遺伝子発現を適応しています。 ここで使用されるティシステムは、ドキシサイクリン（DOX）誘導性転写活性化因子（改善されたリバーステトラサイクリントランスアクチベーター、irtTA、短いたTetA）は、トランスジェニックドライバ行（TetActivator）を含む組織特異的ゲノム調節配列の制御下にあります。 （ 図1A）;第二に、テトラサイクリンオペレーター（TetREのTet応答エレメント）の転写制御下で目的の遺伝子を保有するトランスジェニックレスポンダライン（TetResponder）が必要です。したがって、TetActivatorとTetResponderラインの特定の組み合わせの使用が条件付き組織特異することが可能遺伝子発現のFIC操作。 我々は最近、ゼブラフィッシュテールフィン3を再生成人におけるWnt /β-カテニンシグナル伝達の組織特異的機能を探索するためにテトンシステムを利用してきました。ここで説明したプロトコルでは、フィン再生の研究のために、特に、ゼブラフィッシュにおけるテトンシステムのセットアップおよび使用のための作業の流れを説明します。これは、安定したトランスジェニックTetActivatorとTetResponderラインと胚および成体ゼブラフィッシュにおける導入遺伝子の誘導のためのプロトコルを生成する方法の詳細な手順が含まれています。さらに、我々は、成体ゼブラフィッシュフィンの凍結切片の調製のためのプロトコルを含むフィン再生成における組織特異的遺伝子発現の検証のための技術を説明します。さらに、当社はTetActivator導入遺伝子の設計、遺伝子導入方法の選択、およびTetResponder発現の検出のための考慮事項について説明します。したがって、このプロトコルの全体的な目標は、設計図として機能しますセット·アップ機能ティシステムのゼブラフィッシュの関心の任意の組織に適用することができる条件組織特異的遺伝子発現を達成するために。 我々は、（エンハンサー断片;ワイディンガーラボのプラスミドデータベースはありません1247; 図1B）の短いゲノム調節配列を用いて、安定したTetActivatorラインのI-SceIでまたはTol2の媒介性の発生を可能にするTetActivatorベクトルを作成しました。この構築物は、単純ヘルペスウイルスのVP16トランス活性化ドメイン誘導体3F [irtTAM2（3F）]と融合リバースTetリプレッサードメインのM2突然変異体からなるTetActivatorカセットを含みます。それは、同じオープンリーディングフレームからフルオロフォアAmCyanと共発現されているのでTetActivatorエチレンテトラミン（TETA）の発現を容易に監視することができます。 1比5,6：P2Aペプチドは1で別々のタンパク質としてたTetAとAmCyanの産生をもたらすべきであるリボソームスキッピングを媒介します。構築物はまた、Tのpoylinker 5 &#39;を含みます従来のクローニング方法を用いて、対象のゲノム調節配列の挿入を容易にするetActivatorカセット。 、細菌人工染色体（BAC）に再結合することができる（大きなゲノム領域を含む;さらに、我々は、上記のTetActivatorカセットプラスカナマイシン選択カセットからなる構築物（ 図1C。ワイディンガーラボプラスミドデータベースなし1180）作成しました通常発現パターン）導入遺伝子によって模倣される遺伝子の開始コドンに。両方の構築物は、要求に応じてワイディンガーラボから入手可能です。

<table><tbody><tr><td>Breeding boxes</td><td>Aqua Schwarz</td><td>AquaBox 1</td><td></td></tr><tr><td>Compound fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Confocal microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Cryostat</td><td>e.g., Leica, Thermo-Scientific</td><td>varies with the manufacturer</td><td>for cryosectioning</td></tr><tr><td>4&amp;rsquo;, 6- diamidino-2-phenylinodole (Dapi)</td><td>Sigma-Aldrich</td><td>D9542</td><td>use 1/5,000 in PBS&lt;br/&gt; for visualization of nuclei</td></tr><tr><td>Doxycycline</td><td>Sigma-Aldrich</td><td>D9891</td><td>prepare stocks in 50% EtOH at 50 mg/ml (97 mM)&lt;br/&gt; for TetResponder induction</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148</td><td>4% (w/v) paraformaldehyde in PBS, pH 7.5&lt;br/&gt; for fixation</td></tr><tr><td>1x Phosphat-buffer saline (PBS)</td><td></td><td></td><td>1.7 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 5.2 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 150 mM NaCl, pH 7.5</td></tr><tr><td>1x Phosphat-buffer saline + Tween 20 (PBT)</td><td></td><td></td><td>1x PBS with 0.1% Tween 20</td></tr><tr><td>Superfrost Ultra Plus adhesion microscope slides</td><td>Thermo Scientific&amp;nbsp;</td><td>1014356190</td><td>for collection of tissue sections</td></tr><tr><td>Stereo fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>for fluorescence-based genotyping</td></tr><tr><td>Thermocycler</td><td>e.g., Biorad, Applied Biosystems</td><td>varies with the manufacturer</td><td>for PCR-based genotyping</td></tr><tr><td>Tissue freezing medium (TFM)</td><td>Triangel Biomedical Sciences</td><td>TFM-C</td><td>for embedding of tissue samples</td></tr><tr><td>Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)</td><td>Sigma-Aldrich</td><td>E10521</td><td>for anesthesia&lt;br/&gt; use at 1 mg/ml in E3 embryo medium</td></tr></tbody></table>

use of the teton system to study molecular mechanisms of zebrafish regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

組織特異的な誘導性遺伝子発現、トランスジェニックTetActivatorとTetResponderラインのための機能テトンシステムを確立するために生成された（ 図1A）する必要があります。初期のゼブラフィッシュ胚およびその後の生殖系列の統合に構築またはTetResponder（ 図1E） -これはTetActivator（C図1B）を微量注入することによって達成されます。機能TetActivator構築物の?...

Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

ゼブラフィッシュ再生の分子機構を研究するためにテトンシステムの利用

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

use-teton-system-to-study-molecular-mechanisms-zebrafish

Research

JoVE Journal

Developmental Biology

10.6K ビュー.  Ulm University. Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

ビデオ: Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

ゲンタマイシン誘発性損傷によって大人ゼブラフィッシュにおける腎再生

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

発生生物学

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

タイムラプス明実体顕微鏡でゼブラフィッシュ幼虫における組織修復をキャプチャ

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

ナノ粒子を介した siRNA 遺伝子サイレンシング アダルト ゼブラフィッシュ心臓で

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

幼虫のゼブラフィッシュにおける脊髄切断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

神経科学

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

ゼブラフィッシュ成体終脳の刺し傷傷害：脊椎動物の脳神経発生·再生を調査する方法

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

ゼブラフィッシュモデルの筋肉再生を調べる

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

ゼブラフィッシュとメダカを用いた成体視神経筋の刺創損傷モデルによる再生能力の比較解析

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

<meta charset="utf8"></head><body>再生・開発研究のための堅牢なゼブラフィッシュ細胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>著者スポットライト:発生および再生中のゼブラフィッシュ神経系における細胞接続性のマッピング

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

ゼブラフィッシュの胚および幼生における高分解能細胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

ゼブラフィッシュ再生の分子機構を研究するためにテトンシステムの利用

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この動画の章

ゲンタマイシン誘発性損傷によって大人ゼブラフィッシュにおける腎再生

タイムラプス明実体顕微鏡でゼブラフィッシュ幼虫における組織修復をキャプチャ

ナノ粒子を介した siRNA 遺伝子サイレンシングアダルトゼブラフィッシュ心臓で

幼虫のゼブラフィッシュにおける脊髄切断

ゼブラフィッシュ成体終脳の刺し傷傷害：脊椎動物の脳神経発生·再生を調査する方法

ゼブラフィッシュモデルの筋肉再生を調べる

ゼブラフィッシュとメダカを用いた成体視神経筋の刺創損傷モデルによる再生能力の比較解析

ゼブラフィッシュの胚および幼生における高分解能細胞移植

ゼブラフィッシュの胚および幼生における高分解能細胞移植

ゼブラフィッシュの胚および幼生における高分解能細胞移植

ゼブラフィッシュ再生の分子機構を研究するためにテトンシステムの利用

要約

さらに動画を探す

この動画の章

ゲンタマイシン誘発性損傷によって大人ゼブラフィッシュにおける腎再生

タイムラプス明実体顕微鏡でゼブラフィッシュ幼虫における組織修復をキャプチャ

ナノ粒子を介した siRNA 遺伝子サイレンシング アダルト ゼブラフィッシュ心臓で

幼虫のゼブラフィッシュにおける脊髄切断

ゼブラフィッシュ成体終脳の刺し傷傷害：脊椎動物の脳神経発生·再生を調査する方法

ゼブラフィッシュモデルの筋肉再生を調べる

ゼブラフィッシュとメダカを用いた成体視神経筋の刺創損傷モデルによる再生能力の比較解析

ゼブラフィッシュの胚および幼生における高分解能細胞移植

ゼブラフィッシュの胚および幼生における高分解能細胞移植

ゼブラフィッシュの胚および幼生における高分解能細胞移植

ナノ粒子を介した siRNA 遺伝子サイレンシングアダルトゼブラフィッシュ心臓で