この作品は、ジョンズホプキンス大学でALS研究のためのパッカードセンター（ADG）、NIHのディレクターの新イノベーター賞1DP2OD004417 - 01（ADG）、NIH R01 NS065317（ADG）、リタアレン財団奨学生賞からの助成金によって支えられて。 ADGは、ピュー慈善財団がサポートしている医歯薬学総合研究でピュー学者です。

1。酵母の形質転換のための準備このプロトコルは、10、96ウェルプレート用に設計されていますが、それに応じて上または下に拡張することができます。我々は、このプロトコルは、変換のラウンドあたり20以上の96ウェルプレート用にうまく動作しないことがわかりました。全体の変換手順は（ステップI.3から）約8時間かかります。 <ol><li> BiorobotのRapidPlate液体ハンドラ付き丸底96ウェルプレートの各ウェルに酵母FLEXGene ORFのライブラリーからのアリコットプラスミドDNAの5 -10μL（100 ng /μLの）。場所は乾燥して一晩クリーンベンチドラフト内で96ウェルプレートを発見。我々は、CENと2μ酵母プラスミドと同様の形質転換効率を発見した。 </li><li> 500mLにバッフル付き三角フラスコ内のクエリ株と酵母ペプトンデキストロース（YPD）メディア（10グラム/ L酵母エキス、20グラム/ Lペプトン、20グラム/ Lグルコース）の200mLのを接種する。 （200rpm）し振とうしながら30℃で一晩インキュベートする。必要な場合、選択培地はまた、これらのスターター培養に使用することができます。 </li><li>次の朝は、YPDの2LでOD 600 = 0.1をクエリ菌株を希釈する。 30 ° C（200回転）の文化がOD 600 = 0.8から1.0に達するまでに4〜6時間振とうする。最適な成長のために、独立した2.8 Lバッフル付きフラスコに2つの1Lの文化で育ったの。必要に応じて、選択培地は、YPDの代わりに使用できます。 </li></ol> 2。酵母の変換<ol><li>遠心分離により細胞を回収。卓上遠心機で5分間、3000rpmの（〜1800 × g）で（エッペンドルフ5810R）で酵母培養液とスピンを持つ4つの使い捨ての225mLコニカルチューブを埋める。上清を捨て、すべてのセルが収穫されるまで繰り返す。 </li><li>オートクレーブした蒸留H 2 Oの200mLので細胞を洗浄振ったり、ボルテックスして細胞ペレットを再懸濁します。 one 225mLコニカルチューブに細胞を組み合わせる。 5分間3000rpmの時にスピン。上清を取り除く。 </li><li> 0.1MLiOAc/1XTE溶液（0.1M LiOAc、10mMのトリス、1mMのEDTA、オートクレーブ蒸留水のpH 8）を準備する。 100mLの0.1MLiOAc/1XTEで細胞を洗浄。 5分間3000rpmの時にスピン。上清を取り除く。 </li><li> 70mL 0.1M LiOAc/1xTEで再懸細胞ペレット。ペレットが完全に再懸されていることを確認すると/またはボルテックス振る。 </li><li> 30分（200rpm）し、30℃で振盪しながらインキュベートする。 </li><li> 0.1Mにβ-メルカプトエタノールを追加。 30分（200rpm）し、30℃で振盪しながらインキュベートする。注：β-メルカプトエタノールに敏感な酵母株を使用している場合、このステップは省略することができます。我々は、このステップが成功した変換のために不可欠ではないことを発見した、しかしそれは、形質転換効率を向上させます。 </li><li> 15分間超音波処理したサケ精子DNA（10mg/mL）の2mLのを沸かし、次に氷上で冷やします。 </li><li>細胞混合物にゆでサケ精子DNAの2mLのを追加。注意：沈殿物は、細胞の混合物にサケ精子DNAの添加により形成することがあります。 pipetmanと200μlの先端を使用して、慎重にこれは下流のピペッティングを妨害する可能性があるため、フォームの沈殿物を取り除く。 </li><li> BioRobotのRapidplateを使用して96ウェルプレートの各ウェルに細胞混合物のアリコート50μLは、（また、マルチチャンネルピペットを使用することができます）。混ぜて使用しないでください。 </li><li>振盪せずに30分間室温でインキュベートする。 </li><li> 40パーセントPEG-3350/10％DMSO/0.1M LiOAc（50％PEG - 3350、20mlのDMSO、20mlの1M LiOAcの160mL）の200mLの準備。使用直前に溶液を調製します。 </li><li>各ウェルにPEG / LioAc / DMSO溶液の125μLを追加。 RapidPlateで8回吸引し、細胞懸濁液を分注して混ぜる。 </li><li>振盪せずに30分間室温でインキュベートする。 </li><li>熱ショックで42セル℃で乾燥したインキュベーターで15分間。プレートを積み重ねないでください。注：我々は、この熱ショックのステップが成功した変換のために不可欠ではないことを発見した。特定の酵母菌株（例えば、温度感受性変異株）については、熱ショックは有害です。我々は以前に1分に熱ショック時間を短縮していると正常にこのプロトコルを使用してypt1温度感受性突然変異を保有する酵母株を形質転換することができた。 </li><li> 96ウェルプレートに対応するために遠心アダプターを使用して、5分間2500rpm（〜1100 × g）でプレートを回転させる。廃棄物のバケット上に反転板でのPEG溶液を除去。残液を除去するためにペーパータオル上で逆さプレートをブロット（細胞がウェルの底に残ります）。 </li><li> RapidPlateで各ウェルに（例えばSD / - 裏）最少培地の200μLを加えることによって細胞をすすいでください。 </li><li> 5分間2500rpmで板を回転し、反転以上廃棄物のバケツやペーパータオルでブロッティングにより上清を取り除く。 </li><li> RapidPlateで各ウェルに最少培地の200μL（例えばSD / - 裏）を追加します。 </li><li>振盪せずに2日間、30℃でインキュベートする。 2日後、細胞の小さなコロニーがうまくうまく変換された各の下部にあるフォームを開始する必要があります。 </li></ol> 3。アッセイをスポッティング<ol><li>の各ウェルにラフィノースベースの最少培地（例えばSRAF /浦）の200μLを分注する新しい平底96ウェルプレート。 </li><li> Rapidplateで8回吸引し、細胞懸濁液を分注することによって形質転換プレートの各ウェルを混ぜる。 </li><li>形質転換プレートから細胞混合物の5μLとSRAFプレートに接種する。 </li><li>一日のために30℃でインキュベートする。小さなコロニーは、1日後に各ウェルの底に存在すべきである。 </li><li> SD / -裏とSGal /浦フードのプレート上のスポットのコロニー。炎によって96ボルトレプリケータ（フロッガー）を滅菌する。選択培地プレート上にスポッティングする前にフロッガーを持つコロニーを混ぜる。スポッティング後、プレートを5〜10分間フード内で乾燥させます。 </li><li> 2〜3日間30℃でインキュベートする。 </li></ol>写真デジタルカメラとプレート、そして視覚的にクエリひずみ毒性が増強されるコロニー（遅い成長/密度の低いコロニー）または抑制を（速く成長/より緻密なコロニー）を見つけるためにSGal /浦プレート上のコロニーの成長を比較する。 4。代表的な結果： <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="図1"/> TDP - 43毒性の抑制およびエンハンサーを同定するために図1。酵母プラスミド過剰発現の画面。 TDP - 43は、筋萎縮性側索硬化症（ルーゲーリッグ病）の病因に関与しているヒトタンパ​​ク質である。 TDP - 43の細胞質凝集体は、ALS患者17の脳と脊髄の神経細胞に蓄積されます。凝集と細胞毒性18の酵母細胞の結果におけるTDP - 43を表現する。我々は、TDP - 43毒性19,20のメカニズムを定義するには、このモデルシステムを使用している。示すように私たちの酵母TDP - 43毒性の修飾子画面から板のrepesentative例です。これらのプレートはFLEXGene ORF発現ライブラリーからのプラスミドで形質転換した統合型ガラクトース誘導TDP - 43プラスミドともあるコロニーが表示されます。左側のプレートは、TDP - 43またはFLEXGeneプラスミドの発現を抑制するグルコースを、含まれています。右側のプレートは、TDP - 43の発現とFLEXGeneプラスミドのORFを誘導するガラクトースが含まれています。緑の矢印は、TDP - 43の毒性を抑制することをプラスミドで形質転換コロニーを示している。赤い矢印は、TDP - 43の毒性を増強することをプラスミドで形質転換コロニーを示している。

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利害の衝突は宣言されません。

ここでは、酵母における高スループットプラスミド過剰発現の画面を実行するためのプロトコルを提示する。このアプローチは、多くの異なる細胞の表現型の遺伝的修飾のための迅速かつ公平な審査が可能になります。このアプローチを使用して、研究者は数週間のうちに酵母ゲノムのかなりの部分を選別できます。この公平なアプローチは、以前の調査結果に基づいて予測されていない可?...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td> BioRobot RapidPlate </td><td>キアゲン</td><td> 9000490 </td></tr><tr><td> 96ボルトレプリケータ（フロッガー） </td><td> V＆Pサイエンティフィック</td><td> VP404 </td></tr><tr><td> FLEXGene ORF図書館</td><td>プロテオミクスの研究所、ハーバードメディカルスクール</td><td></td></tr><tr><td>卓上遠心機</td><td>エッペンドルフ</td><td> 5810R </td></tr><tr><td> 500mLにバッフルフラスコ</td><td> Bellco </td><td> 2543〜00500 </td></tr><tr><td> 2.8Lトリプルバッフルフェルンバッハのフラスコ</td><td> Bellco </td><td> 2551〜02800 </td></tr><tr><td> 100μLRapidplateピペットチップ</td><td> Axygen </td><td> ZT - 100 - RS </td></tr><tr><td> 200μLRapidplateピペットチップ</td><td> Axygen </td><td> ZT - 200 - RS </td></tr></tbody></table>

high-throughput yeast plasmid overexpression screen

出芽酵母、 サッカロマイセスセレビシエは 、ヒトの疾患への直接的な関連性を有するものを含む多くの重要な細胞プロセス、の基本的なメカニズムを定義するための強力なモデルシステムです。で、短い世代時間と十分に特徴付けゲノムから、酵母のモデルシステムの主要experimental利点は、特定のプロセスに関与する遺伝子と経路を同定する遺伝子スクリーニングを実行する機能です。 30年間にわたってこのような遺伝子スクリーニングは、細胞周期、分泌経路、および真核細胞生物学1-5の多くの高度に保存された側面を解明するために使用されている。ここ数年では、酵母菌株とプラスミドのいくつかのゲノムワイドライブラリは6-10生成されている。これらのコレクションは、現在の利得と機能喪失型のアプローチ11-16を使用して遺伝子機能の系統的な尋問を可能にする。ここでは、5500酵母プラスミドのアレイライブラリを使用して、プラスミド過剰発現の画面を実行するために液体ハンドリングロボットによるハイスループット酵母の形質転換プロトコルを使用するための詳細なプロトコルを提供しています。我々は、凝集が発生しやすい人間の神経変性疾患のタンパク質の蓄積に関連付けられている毒性の遺伝的修飾を識別するために、これらの画面を使用している。ここで紹介する手法は、関心のある他の細胞の表現型の研究に迅速に適用可能であり。

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

ここではでプラスミド過剰発現の画面を説明<em&gt;サッカロマイセスセレビシエ</em&gt;、アレイプラスミドライブラリーと液体ハンドリングロボットによるハイスループット酵母の形質転換プロトコルを使用して。

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, ...

high-throughput-yeast-plasmid-overexpression-screen

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JoVE Journal

Biology

16.3K ビュー.  University of Pennsylvania School of Medicine. ここではでプラスミド過剰発現の画面を説明サッカロマイセスセレビシエ、アレイプラスミドライブラリーと液体ハンドリングロボットによるハイスループット酵母の形質転換プロトコルを使用して。

ビデオ: High-throughput Yeast Plasmid Overexpression Screen

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

メタゲノムライブラリからセルラーゼ活性をBiominingためのハイスループットスクリーニング

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

生物学

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 ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

アフィニティータグ組換えタンパク質のハイスループット精製

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

ハイスループットSHAPEを使用したRNA二次構造予測

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

ウニ​​接合体のハイスループットマイクロインジェクション

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

ルシフェラーゼを発現する組換えウイルスの高スループット滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

ゼブラフィッシュ胚を用いた高スループットへのアプローチマニュアル小分子スクリーン

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

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 ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

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 ...