The authors would like to thank Drs. Roger Cone and Chao Zhang for their contributions to validating the application of this assay as previously published. This work was supported by NIH 1F32DK082167-01 (BJR).

注：このプロトコルは、研究の責任ある行動のためのアリゾナ大学の事務所のガイドラインに従っており、施設内動物管理使用委員会によって承認されました 1.ゼブラフィッシュの繁殖や胚のメンテナンス<ol><li> E3胚媒体 8 を準備します。 60Xストック溶液を作るために、1.95 L ddiH 2 O中に34.8グラムのNaCl、1.6グラムのKCl、5.8グラムの塩化カルシウム2●2H 2 O、および9.78グラムのMgCl 2●6H 2 Oを溶解ddiH 2 Oを使用して、2リットルにボリュームを調節NaOHおよびHClを用いてpHを7.2に調整します</li><li> 1倍のE3胚の培地を調製するために、1 L ddiH20に16.5ミリリットル60倍株式を希釈します。 100μlの1％のメチレンブルーを追加します。 </li><li>育種<ol><li>ハウス男性と女性ひな株式（7 - 生後18ヶ月）を別々に繁殖力を最大限に高めることができます。 </li><li>卵が所望される前日に前に2時間 - 1からライトをオンにします。 C言語で - セットアップ交配（2人の女性1男性と1）以前に9に記載されているようにケージをrossing。彼らは大人の魚は保持されたタンクインサートの穴あきベースを通過する卵が捕食を逃れるためにするための横断ケージができます。 </li><li>オプション：時限繁殖のために、（モルホリノ注入を含む研究のために必要とされるように）、透明なプラスチックの分周器によって、オスとメスの魚を分離し、卵と受精のタイミング敷設をもたらすことが午前中にこれを削除してください。モルホリノ研究のために、治療ごとに必要な魚の胚の数を評価するためにパワー計算を実行します。効果や変動の大きさの推定値がなければ、予備試験では、N = 8で実行することができます。 </li></ol></li><li> （ - 150胚/ 10cmのペトリ皿100）ペトリ皿に交差ケージから卵を注ぎます。卵は皿の底に沈殿し、水を注ぎ出すようにします。先端の細い使い捨てのトランスファーピペットを用いて、残りの水を除去します。ステップ1.1で行われたE3の胚培地を追加し直します。 </li><li> AFを作ります将来卵や魚転送用のラメポリッシュ使い捨てピペット。はさみを使用して、0.25ミリリットルの卒業で卒業使い捨てトランスファーピペットをカット。ラフなエッジを削除するには、炎はブンゼンバーナーの上にカットチップを磨きます。 </li><li>ゼブラフィッシュのE3胚の培地中のペトリ皿28.5○Cで受精後10センチメートル中（DPF）4日に胚魚を維持1日2回、洗練された火炎を使用するすべての未受精卵または死ん胚（不透明な白い外観）を除去するために使い捨てのトランスファーピペットを卒業。 1日に1回、E3胚培地を交換してください。 E3の胚培地を捨て、先端の細い使い捨てのトランスファーピペットを使用して残りの液体を除去し、予め温めた（28.5○C）のE3胚培地を再度追加。 </li></ol> 2.アッセイ溶液を調製<ol><li> 0.22μmのフィルターを使用して、表1の無菌フィルターアッセイ溶液によるアッセイ溶液を調製します。アッセイ開始前に、28.5○Cで滅菌アッセイ溶液を温めます水浴。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td>成分</td><td>合計の％ </td><td>容量（μL/ 10ミリリットル） </td></tr><tr><td> 60X E3胚培地</td><td> 1.667 </td><td> 166.7 </td></tr><tr><td>二重脱イオン（DDI）、H 2 O </td><td> 96.233 </td><td> 9623.3 </td></tr><tr><td>アラマーブルー</td><td> 1 </td><td> 100 </td></tr><tr><td> 400mMのNaOHを</td><td> 1 </td><td> 100 </td></tr><tr><td> DMSO </td><td> 0.1 </td><td> 10 </td></tr></tbody></table> 表1アッセイ培地 3.アッセイを行います<ol><li>ゼブラフィッシュの胚を滅菌濾過したのE3胚培地で3回洗浄します。クラッチからのすべての実行可能なゼブラフィッシュの胚をビーカーに結合します。滅菌フィルタ75ミリリットルのE3胚培地。先端の細い使い捨てトランスファーピペット、再使用できるだけゼブラフィッシュ胚からのE3胚培地の多くを移動します。 20ミリリットルに戻って濾過滅菌のE3胚培地を追加します。削除およびE3胚培地を3回交換してください。 </li><li> 96ウェル黒色の93ウェルの各々にプレート1魚は明確な底板を両面。個別に炎の中に胚を捕捉することにより、プレートに胚魚を移し卒業プラスチック使い捨てトランスファーピペット（ステップ1.5）を研磨し、優しくウェルに放出します。 3ブランクウェルにすすぎ、魚の入ったビーカーから取り出したのE3胚培地を転送します。これは、ブランクウェルには魚が含まれている井戸とまったく同じ水にさらされていることを保証します。魚やブランクウェルを含める両方ウェルは、このステップの終了時（200μl）を1/2以上完全でなければなりません。 </li><li>ブランクウェルを含む96ウェル全てにおいて、アッセイ溶液でのE3胚培地を交換してください。このステップは、96ウェルプレート中12各列の時に1列を実行する必要があります。先端の細い使い捨てのトランスを使用しましたFERピペットは、プレートの1列（8ウエル）ですべてのウェルからのE3胚培地を除去します。ゼブラフィッシュの胚には手を触れないように注意してください。水を除去しただけでなく、そこからそれぞれにアッセイ溶液を300μlを追加します。プレートの全12列のためのアッセイ溶液でのE3胚培地と交換の除去を繰り返します。 </li><li>オプション：化合物に応答した代謝率の変更をテストするには、100倍（100X）所望の治療濃度で溶液を作製。注目すべきは、100X溶液は0.1％を超えないように、最終DMSO濃度を制限するために10％のDMSOを超えないことを保証します。治療の各ウェルに100倍液の3μLを追加します。 注：処理ウェルの数を評価するために、電力の計算を実行します。しかし、応答または変動の大きさの推定値なしに、予備試験は8化合物で処理し、魚が含まれている8ビヒクル処置対照ウェルで実行することができます。 <ol><li> （コントロールに何のDRUを車両制御の3μlを添加していませんグラム処理）魚が含まれている井戸。化合物に対する蛍光応答が魚内のアクションの結果であることを保証するために、96ウェルプレートに3ブランクウェルに薬物および車両の治療を適用します。 </li></ol></li><li> 590 nmで530 nmでの励起波長および発光波長で蛍光プレートリーダー上の魚満たさプレートをお読みください。 </li><li> 28.5○C（アッセイ溶液の光退色を避けるために、アッセイ全体の暗闇の中でプレートを維持する）に設定加湿インキュベーターに置きプレート。アッセイは、1時間から24時間の範囲の時間の持続時間のために実行することができます。アッセイの持続時間は、研究の目的に依存します。短い期間が最も適切である短時間作用型非安定な化合物への応答をテストするため。しかし、安定した化合物の検査や遺伝的影響のテストのために、分析時間を最大にすることの累積信号に関連する利点を活用することが好ましいです。 </li><li>所定の時間点で（タイミングを判断するためにステップ3.6参照）ステップ3.5と同じ設定で、再び魚で満たされたプレートの蛍光を読み取ります。 </li></ol> 4.治療と蛍光関連の相対的変化を評価します<ol><li>研究の結論での蛍光から時間0での蛍光を差し引くことにより、各ウェルの蛍光の変化を計算します。魚ないウェル（ブランク）はるかに魚が含まれているウェルからの値の下に、0に近い値を持つ必要があります。 時間0での蛍光 - 終了時に蛍光=蛍光の変化</li><li>対照ウェルは、その後、コントロールウェルの蛍光の平均変化によって、各ウェルの蛍光の変化を分割するための蛍光の相対的変化を確認するために、蛍光の平均変化を計算します。注目すべきは、コントロールは、車両コントロールまたは遺伝的コントロールのどちらかである可能性があります。 <img alt="式（1）" src="/files/ftp_upload/53297/53297eq1.jpg" /></li></ol>

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The authors have no competing financial interests to disclose.

細菌や真菌の混入が大幅にこのアッセイの適用を制限します。 E3胚培地中でメチレンブルーは、真菌汚染の可能性を制限します。胚全体に飼育ケアは毎日二度死んだ胚を除去するために取られることが重要です。加えて、徹底的に分析開始の日に、無菌のE3胚培地で胚を洗浄することが不可欠です。これらの2つのステップは、胚の細菌汚染の可能性を制限します。ステップ3.2に記載のブラン?...

マウスは現在、肥満研究のための主要なモデルです。ショート発生間隔とマウスで利用可能な遺伝子ツールは、これまでに比類のないされています。しかし、ゼブラフィッシュも短く発生間隔があり（3から4ヶ月）と遺伝子操作1,2の容易さであっても、マウスを凌駕しています。これまでの遺伝子と薬剤スクリーニング3で使用するための子孫と潜在的な数でマウスを越えるとゼブラフィッシュは、哺乳動物遺伝子の約90％を維持しています。 肥満研究のためのゼブラフィッシュモデルの可能性をタップするには、アッセイは、エネルギー消費を含む体重調節に影響を与える要因を調査するために開発されなければなりません。代謝産物は、β酸化によって処理され、トリカルボン酸サイクルの酸素が ​​消費され、NADH 2が生成されます。このように、NADH 2は、代謝経路（代謝物の酸化）を介して、代謝物のフラックスの直接的な指標です。 poikilで恒温動物4に比べて5倍低い- otherms、H + ATP合成からNADH 2酸化を脱共役ミトコンドリア内膜、漏れるは、4です。したがって、2ゼブラフィッシュNADHには非常にしっかりと酸化的リン酸化を介してATP産生にリンクされています。ここで、我々は、エネルギー消費5のプロキシとして幼虫のゼブラフィッシュにおけるNADH 2産生を測定するアッセイを説明します。 酸素消費量は、エネルギー消費を測定するためのゴールドスタンダードです。しかし、最高のゼブラフィッシュの高スループットの可能性を活用するために、エネルギー消費のアッセイは、ハイスループットに従順でなければなりません。密閉チャンバー循環系に依存酸素消費システムは、利用可能なチャンバ6の数でスループットが制限されます。外気O 2消費量/ CO 2産生のアッセイはまた、ゼブラフィッシュ5,7に適用されています。これらのオープンエアの96ウェルプレートベースDシステムは、ハイスループットに適しています。残念ながら、環境とのガス交換は、これらのアッセイの感度を制限します。我々は最近、酸化還元指示薬アラマーブルー5を使用して、NADH 2をモニターするアッセイのアプリケーションを発表しました。このアッセイは、スループットおよびゼブラフィッシュにおける酸素消費量の分析に共通の感度の限界を克服します。 ゼブラフィッシュは、全身のエネルギー恒常性の研究のためにますます重要なモデルとなってきています。一部では、ゼブラフィッシュため、前方の遺伝子スクリーニングおよび薬物スクリーニングでの使用に適しています。また、ノックダウンとノックインを含む遺伝子操作を、ターゲットに、迅速に適用することができます。我々は以前に、このアッセイは、化合物および代謝率5を変更する遺伝子を同定するためにバス薬物適用および遺伝子ノックダウンまたはノックアウトと組み合わせることができることを示しています。さらに、このアッセイは、ゼブラフィッシュに固有の高スループットの利点を活用するように設計されています。 </P&gt;

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">AlamarBlue</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">10-230-103</td>
			<td style="width:307px;">Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fine Tip DisposableTransfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-711-26</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Fisherbrand Graduated Disposable Transfer Pipette</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td>13-771-9AM</td>
			<td>Manufactured by Thermo Scientific</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Black sided clear bottom 96-well plates</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">50-320-785</td>
			<td>Manufactured by E&#38;K Scientific Products Inc.</td>
		</tr>
	</tbody>
</table>

high throughput danio rerio energy expenditure assay

ゼブラフィッシュは、ゼブラフィッシュエネルギー恒常性と肥満の研究に広く適用モデルにする可能性を持っている固有の利点と重要なモデル生物です。ゼブラフィッシュの小さなサイズは、96または384ウェルプレート形式で実施される胚でのアッセイを可能にする、モルホリノ及びCRISPRベースの技術は、バス・アプリケーションによって遺伝子操作の容易性、および薬物治療を促進する実行可能です。また、ゼブラフィッシュは、新規遺伝子の発見を可能にし、前方の遺伝子スクリーニングに最適です。肥満のモデルとしてゼブラフィッシュの相対的新規性を考えると、それは完全にこれらの利点を活用するツールを開発することが必要です。ここで、我々は同時に、胚ゼブラフィッシュの数千にエネルギー消費を測定するための方法が記載されています。我々は個々の魚を隔離するために96ウェルプレートを使用した全動物のマイクロプレートプラットフォームを開発していると我々は、市販の細胞培養の生存率の再を使用して、累積NADH 2産生を評価しますエージェントアラマーブルー。 poikilothermsでNADH 2生産とエネルギー消費の関係が緊密にリンクされています。このエネルギー消費アッセイは急速に直接エネルギー消費を変更するか、適用された薬剤（例えば、インスリン増感剤）に対する応答を変化させる薬理学的または遺伝子操作をスクリーニングする可能性が作成されます。

アッセイ溶液は、胚魚（ 図1）の非存在下で蛍光を増加させません。しかし、このアッセイは、ウェル内の代謝率の小さな変化に非常に敏感です。ウェル内の単一の魚は1時間（P &lt;0.0001）内のブランクウェルと著しく異なる信号を生成する。 図2は、インキュベーションの24時間後に胚魚によって生成された信号を視覚的に表現しています。この写真の目的のために?...

Watch this Scientific Journal Video about High Throughput Danio Rerio Energy Expenditure Assay at JoVE.com

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高スループット<em&gt;ゼブラフィッシュ</em&gt;エネルギー消費アッセイ

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

high-throughput-danio-rerio-energy-expenditure-assay

Research

JoVE Journal

Biology

High Throughput Danio Rerio Energy Expenditure Assay

12.6K ビュー.  Vanderbilt University Medical Center. Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery. 

ビデオ: High Throughput Danio Rerio Energy Expenditure Assay

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

アダルトゼブラフィッシュの内耳感覚上皮のグロスとファイン解剖（ゼブラフィッシュ）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

生物学

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

ハイスループット酵母プラスミドの過剰発現の画面

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

リグノセルロース系材料のためのハイスループット糖化アッセイ

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

ゼブラフィッシュの定期的ケアとメンテナンス（<em&gt;ゼブラフィッシュ</em&gt;）研究所：はじめに

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

ゼブラフィッシュを用いた病的ヒトゲノムの in vivoモデルで

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

ペプチドエピトープの定量分析のための高スループット、MHC II結合アッセイ

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFEアッセイを用いた高スループットにおけるmiRNA標的の検出

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

高スループット

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