我々は、ゼブラフィッシュの飼育に彼女のサポートのためのさんファンシェンに感謝。これは、博士ビング胡（中国の科学アカデミー、&quot;白仁智華&quot;、NSFC 30870833と中国の国家基礎科学プログラム、2009CB941302）へのファンディングでサポートされています。 全ての動物実験は、科学と中国（USTC）動物資源センターと大学動物実験委員会の技術の大学が定めるガイドラインおよび規制に準拠して実施された。

パートI：OKRの装置を構築詳細は、ビデオのデモで示されています。 パートII：大人ゼブラフィッシュのOKR ラボがOKR装置を構​​築している場合は、成人ゼブラフィッシュOKRの鍵は、魚を保持することです。このパートでは、私たちは、あなたがステップバイステップで紹介します。 私たちの装置は、前述の他の人と同様の以下の部分を構成している。円形の魚槽が外径7.0センチ、内径は6.5センチ、高7.0 cmです。魚槽の周りドラムは外直径は8.5センチ、内径は7.5センチ、高7.0 cmです。それは時計回りに20〜スピード〜分当たり70回転（rpm）と反時計回りに回転させることができます。黒と白のストライプの11サイクルは、ドラムの外側に付着したトレーシングペーパーに描かれています。光システムは、21 cmの直径の2つの円形の光のチューブで構成されています。 <img src="/files/ftp_upload/1742/1742fig1.jpg" alt="図1" /> 図1。スポンジのモデルは、この真ん中に作られた崖は、視野が守られていなかったことを確認することです。 <ol><li>図1に示すようにスポンジは二つの部分により構成。左側の部分は長さ3センチメートル×幅3センチ× HIGH 2 cmです。右側の部分は長さ3センチメートル×幅3センチ× 3上位cmです。 </li><li>魚槽の底にこのスポンジを入れ、約2 cmの深さで飼育水を注ぐ。スポンジで非表示の気泡が追い出されるはず。 </li><li>崖の背後にある約2mm、スポンジの右側の部分の前にピンを挿入します。 </li><li> MS - 222の大人のゼブラフィッシュを麻酔。 </li><li>背上にスポンジにゼブラフィッシュを入れて、最初のピンの前にその胸びれの片側を許可する。 </li><li>つ目のピンは、他の胸びれの後ろに挿入されます。それは、最初のピン（これも図2参照）との連携によりゼブラフィッシュ縮小ボディを避けることができます。 </li><li>動脈と尾をクランプし、魚槽に付着しているゴムの部分（粘土）の動脈のクランプをクランプして固定する。 </li><li> 3番目と4番目のピンは、魚の体の総排泄孔の近くのサイトに植えられている。それは、激しく尾を振ってゼブラフィッシュを防ぐことができる。 </li><li>番ピンが6番目のピンはそれの反対側にあるし1番目と3番目のピンとの間で土地を選定される。彼らは、その本体を振ってからゼブラフィッシュを防ぐことができます。 </li><li> 7番目と8番目のピンはヘッドの向きを保つために鰓に近い部位に挿入されます。 </li><li>魚室内の飼育水を注ぐ。 </li><li>ゼブラフィッシュは、ウェイクアップ後に、それは5〜10分間に適応できます。 </li><li>この装置の光のスイッチを入れ、ゼブラフィッシュは、2分間この照明を適応させることができます。 </li><li>回折格子を駆動し、1分間に適応するためにゼブラフィッシュを許可するモーターのスイッチをオンにします。 </li><li>カメラの電源をオンにし、目の動きを記録。 </li><li>パーソナルコンピュータ（眼角の解析の代表的な例を図2に示されている）で目の角を6とサッカードの数字を分析する。 </li></ol><img src="/files/ftp_upload/1742/1742fig2.jpg" alt="図2" /> 図2。眼軸と体軸との間の角度。 a）は格子の方向は、鼻から頭の場合は、我々は、魚の体の前方に角度を定義し、負の角度であり、魚の体の角度は、正の角度、その逆（簡単なレビューのためにも文献を参照してくださいです。 6）。この図中の数字はピンのシーケンスを表します。 通常のグループ内の1つの魚からb）のOKRの解析例。一第2作は、シリーズのフレームに移し、各フレームの目の角度はイメージ- PRO Plusソフトウェアを用いて測定した。この図の各点は、我々が測定した各フレームを表します。 注意事項： <ol class="lalpha"><li>すべてのピンは単にその本体を振ってからゼブラフィッシュを防止するために使用されている、あまりにもタイトな魚に有害になります。 </li><li>その接触面積は小さいものですので動脈のクランプは、尾を損傷する恐れがあります。尾がフリーになると、それはこの魚を修正することは非常に困難S。いくつかの条件で推奨されている尾の周り包帯を巻きます。 </li><li>鰓に近いサイトに植えられている7番目と8番ピンは格子の彼らの姿から避難すべきではない。 </li></ol>パートIII。幼生ゼブラフィッシュのOKR 1。メチルセルロース長い間生き続ける間、幼虫のゼブラフィッシュは6％メチルセルロース溶液で固定化することができる。次のようにメチルセルロース溶液を準備します。 <ol><li>ハイレベルで約3分電子レンジで沸騰200ミリリットルのddH 2 O。 </li><li>攪拌しながらホットプレートにお湯を移し、精力的にそれをかき混ぜる。 </li><li>徐々にお湯に12gのメチルセルロースを追加。 </li><li>メチルセルロースはよく分散された後、3分間攪拌してください。 </li><li>それが透明になるまで氷水でソリューションを冷却し、4℃に置きます℃に</li></ol><pクラス=&quot;jove_step&quot;&gt; 2。幼虫のゼブラフィッシュ<ol><li>ペア二人の男性と授乳後の17:00交配タンク内の成人ゼブラフィッシュのメス。翌朝は、新鮮な交配のタンクにそれらを転送し、1時間後に胚を収集する。 </li><li> 14時10分の暗サイクル：卵を光で28.5 ± 0.5℃のインキュベーターで胚の培地中に維持されています。 </li><li>卵は48時間後に受精（HPF）で行わ孵化され、OKRは後で72 HPFまたはでテストされます。このビデオでは、larvaes 5 DPFにあります。 </li></ol> 3。 OKRの手順<ol><li>この装置の光をCCDカメラとスイッチを装備したステレオスコープの下に装置を置く。 </li><li> 55 mmのペトリ皿に20ミリリットル6％methycelluloseを追加。 </li><li>この皿の中央にある行でいくつかのlarvaesを置きます。 </li><li>カメラのフィールドに1μlの約気泡を入れて、それは格子の方向を反​​映する鏡として可能性があります。 </li><li>モーターのスイッチを入れ、彼らの目の動きを記録。 </li></ol>パートIV。アダルトゼブラフィッシュのOMR 成人ゼブラフィッシュOMRもこの装置で実施することが、ちょうど（回転速度は黒 - 白のサイクルが11の36回転、つまり、Fig.3aに示した）センター全体で泳ぐ魚を防止するために魚のチャンバー内に列を配置します。 <ol><li>この魚のチャンバー内の魚を置く。 </li><li>この魚室の中央に2cmの直径のカラムをスタンド。 </li><li>両方まだ格子と回転格子で1分のために適合させる。 </li><li>ゼブラフィッシュの動きを記録します。 </li><li>パーソナルコンピュータの時間の一致率を分析する（例は図3bに示されている、回転速度は36 rpmの場合、白黒サイクルは11です） </li></ol><img src="/files/ftp_upload/1742/1742fig3.jpg" alt="図3" /> 図3。アダルトゼブラフィッシュのOMR。 （a）の白のプラスチックの列は、それ全体で泳ぐ魚を防止するための魚室の中央に置かれた。 （b）の成人ゼブラフィッシュの一致率は格子に続く。偶然の一致率は、総録音時間は以下の格子の時間の割合と定義した。 50％の値は100％が非常に良く魚の以下の格子を示しながら、魚がランダムに泳いでいることを示しています。両方の視神経が約10秒の宝石鉗子で粉砕された。 OMRの動作は、クラッシュ後にランダム水泳として登場。

<ol>
	<li>Beck, J., Gilland, E., Tank, D., Baker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+Ontogeny+of+Optokinetic+and+Vestibuloocular+Behaviors+in+Zebrafish+Medaka,+and+Goldfish.">Quantifying the Ontogeny of Optokinetic and Vestibuloocular Behaviors in Zebrafish Medaka, and Goldfish.</a> J Neurophysiol. 92, 3546-3561 (2004).</li><li>Brockerhoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+optokinetic+response+of+zebrafish+larvae.">Measuring the optokinetic response of zebrafish larvae.</a> Nature Protocols. 1, 2448-2451 (2006).</li><li>Emran, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OFF+ganglion+cells+cannot+drive+the+optokinetic+reflex+in+zebrafish.">OFF ganglion cells cannot drive the optokinetic reflex in zebrafish.</a> Proc Natl Acad Sci U S A. 104, 19126-19131 (2007).</li><li>Rinner, O., Rick, J., Neuhauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+Sensitivity,+Spatial+and+Temporal+Tuning+of+the+Larval+Zebrafish+Optokinetic+Response.">Contrast Sensitivity, Spatial and Temporal Tuning of the Larval Zebrafish Optokinetic Response.</a> Investigative Ophthalmology and Visual Science. 46, 137-142 (2005).</li><li>Roeser, T., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+Behaviors+in+Larval+Zebrafish+after+GFP-Guided+Laser+Ablation+of+the+Optic+Tectum.">Visuomotor Behaviors in Larval Zebrafish after GFP-Guided Laser Ablation of the Optic Tectum.</a> The Journal of Neuroscience. 23, 3726-3734 (2003).</li><li>Easter, S., Nicola, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+of+Vision+in+the+Zebrafish+(Danio+rerio).">The Development of Vision in the Zebrafish (Danio rerio).</a> Developmental Biology. 180, 646-663 (1996).</li><li>Maaswinkel, H., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+frequency+characteristics+of+the+optomotor+response+in+zebrafish.">Spatio-temporal frequency characteristics of the optomotor response in zebrafish.</a> Vision Research. 43, 21-30 (2003).</li></ol>

OKRの定義によれば、それは動物が自発的に回転する格子を追求する彼らの目を移動する現象である。この動作は長い時間（6時間上記の例）のために実施されている場合は、目の筋肉が疲れてかもしれないとOKRは減衰されます。 成人のゼブラフィッシュOMRは他人7によって完全に記述されている。このビデオでは、私達はちょうど私達の装置を使用して、この動作...

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using the optokinetic response to study visual function of zebrafish

視運動反応（OKR）は、動物がその周りに回転する格子に従うことに目を振動する動作です。それは広く幼虫ゼブラフィッシュ1-5の視覚機能を評価するために使用されています。それにもかかわらず、仔魚のための標準プロトコルは、まだ成人zabrafishに容易に適用されません。ここで、我々の研究室で確立される新しいプロトコルを使用して簡単な特注の装置を成人ゼブラフィッシュのOKRを測定する方法を紹介します。私たちの装置と成人のゼブラフィッシュのOKRのステップバイステップの手順の両方がこのビデオに示されています。さらに、幼虫のOKRの測定だけでなく、大人のゼブラフィッシュの微細運動応答（OMR）試験は、またこのビデオでは実証されています。我々の実験における成人のゼブラフィッシュのこのOKRアッセイは、最大4時間まで続くことがある。成魚のビジョンシステムが操作されると、成魚に適用されるようなOKRのテストは、より効率的に視覚機能を調査するために利益になる。 蘇チーゾウと呉陰はこの論文にも同様に貢献した。

Watch this Scientific Journal Video about Using the optokinetic response to study visual function of zebrafish at JoVE.com

Using the optokinetic response to study visual function of zebrafish

視運動性応答は、広く幼虫ゼブラフィッシュの視覚機能を評価するために使用されています。それにもかかわらず、仔魚のための標準プロトコルは、まだ大人に容易に​​適用されていません<sup&gt; 1月5日</sup&gt;。ここで、我々の研究室で確立される新しいプロトコルを使用して、成人ゼブラフィッシュのOKRを測定する方法を紹介します。

ゼブラフィッシュの視覚機能を研究するために視運動性応答を使用して

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

using-the-optokinetic-response-to-study-visual-function-of-zebrafish

Research

JoVE Journal

Biology

18.3K ビュー.  University of Science and Technology of China (USTC). 視運動性応答は、広く幼虫ゼブラフィッシュの視覚機能を評価するために使用されています。それにもかかわらず、仔魚のための標準プロトコルは、まだ大人に容易に​​適用されていません 1月5日。ここで、我々の研究室で確立される新しいプロトコルを使用して、成人ゼブラフィッシュのOKRを測定する方法を紹介します。

ビデオ: Using the optokinetic response to study visual function of zebrafish

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

コンピュータによって生成された動物モデルの刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

遺伝子機能を解析するためにゼブラフィッシュ胚のマイクロインジェクション

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

ゼブラフィッシュ上顎バーベルの研究のための方法

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

ハイC：ゲノムの三次元構造を研究する方法。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

腎上皮再生を研究するためにゼブラフィッシュの前腎のレーザーアブレーション

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

ゼブラフィッシュ幼生における筋機能を評価するために、収縮時の力測定

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解像度全マウント<em&gt;その場で</em遺伝子発現と機能を研究するためにゼブラフィッシュ胚内&gt;ハイブリダイゼーション

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

大人のゼブラフィッシュの腎臓におけるネフロン構成と機能の解析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...