1）ゼブラフィッシュの卵コレクション<ol><li>前化学画面の日に午後に、10〜20ゼブラフィッシュの繁殖タンクを設置。水産養殖システムから水を各タンクを埋める。魚のネットを使用して、それぞれの繁殖水槽の内側の容器に大人一人の男性と一から二成人女性を転送する。分圧器を使用して、お互いからオスとメスの魚を区切ります。ケージにラベルを付け、それらの上に蓋をする。 </li><li>画面の朝に、繁殖タンクから仕切りを削除し、チームメイトへのゼブラフィッシュができます。次の1時間のコースで、受精卵がそれぞれ内側の容器の下部にグリッドフォールスルーすることができます。 </li><li> 1時間後、、永久的な貯蔵タンクに大人のゼブラフィッシュを戻す内側のコンテナを削除し、プラスチック製の茶こしを通して各飼育槽内の水に負担をかけることにより卵を集める。 </li><li>ペトリ皿上のストレーナーを反転し、E3培地を含む洗浄瓶を使用してペトリ皿に卵をフラッシュするように優しくストレーナーをすすいでください。 </li><li>不透明な表示されるすべての未受精卵は、、使い捨てのプラスチック製のピペットを使用して削除する必要があります。各交配のクロスは、約200胚が得られるはず。 </li></ol> 2）96ウェルプレートで胚をアレー<ol><li>ガラスパスツールピペットを用いて96ウェルプレートの各ウェルにE3培地で5胚についての転送。 </li><li>穿刺に胚を気にしないながら、ピペット - 一度胚は同じくらいの12チャンネル（300μL30）を用いて井戸の可能な外としてE3培地のを削除し、96ウェルプレートに配列される。 12チャンネルピペットを使用して、そのような胚が干上がることがないようにしても、できるだけ早く各0.5μg/mlカナマイシンを含むE3培地250μLを提供。 </li><li>化合物を添加するときに必要な段階に達するまで、28.5 ° Cのインキュベーターに96 - ウェルプレートを置く。 </li></ol> 3）小分子ライブラリーの移転は、 化合物の転送がロボットの転送方法を自動化することができますが、我々は、手動転送方法を説明します。 <ol><li>小分子ライブラリーは、典型的には10 mMストックとしてDMSOに格納された各化合物を、96ウェルフォーマットで供給されます。化合物が追加されるときに胚がステージに到達する約60分前に、小分子のアリコート（ソースプレート）を含む96ウェルプレートの希望番号を解凍。ソースプレートのシリアルまたはその他の識別番号をメモしておいてください。プレート上の結露を最小限に抑えるために、融解はDRIERITEを（ワシントン州ハモンドDRIERITE CO、セニア、オハイオ州）を含む乾燥室内で発生する可能性があります。 </li><li>簡単に言うと、マルチウェルプレートアダプタを搭載した卓上型遠心機のプレートをスピンダウン。 </li><li>ソースプレートからアルミシールテープを取り外します。 12チャンネルピペットを使用して、0.5mMの濃度（例えば、10mmの株式250 NLのアリコートで始まる場合、各ウェルにDMSO 4.75μLを加える）にソースプレート内の化合物を希釈する。 </li><li> 96ウェルプレート（受信者のプレート）の胚は、含まれている受信者のプレートにソースプレートから化合物（0.5）の2.5μLを転送するために、12チャンネル（2-20μL）ピペットを使用して、必要な段階に到達したとき胚。 </li><li>レシピエント胚のプレート上にソースプレートの識別番号を記録します。今蓋つきの胚と化合物を含む受信者のプレートをカバーし、穏やかに穏やかに渦巻くことでプレートを混合し、28.5 ° Cのインキュベーターに置いてください。 </li><li>長期保存のため-80℃の冷凍庫にアルミシールテープと場所で使用されていない小分子（0.5 mm）を含む各ソースプレートをカバーしています。 </li></ol>表現型の目視検査による低分子の効果4）スクリーニング<ol><li>画面を実行する前に、&quot;ヒット&quot;を構成するであろう何のために特定の基準を策定する。 </li><li>開発の目的の時間に、インキュベーターから化合物で処理した胚を含む96ウェルプレートを取り出して、実体顕微鏡下で各ウェルを調べます。このような循環パターンの変化などの微妙な変化、のよりよい視覚化のために、位相差倒立顕微鏡を使用することができます。蛍光顕微鏡は、組織特異的プロモーター下にGFPまたはDsRedタンパク質の発現の摂動を調べるために使用することができます。 </li><li>すぐに5胚の内少なくとも3つの任意ウェルの96ウェルプレートは、所定の&quot;ヒット&quot;表現型を示すスキャンします。プレートの識別と各潜在的なヒットのも場所を記録します。 </li><li>いくつかの投与量（1μMの、5μmの、10μMおよび50μM）における化合物の効果を再検査することにより、潜在的なヒットを再確認する。各用量については、10胚を48ウェルプレートフォーマットでのE3メディアの0.5mlにテストされています。再試験のための化合物の添加時期は、オリジナルのスクリーニングのと同じである必要があります。引き出した表現型がcompounの再検査で再現されたときにヒットが確認されD </li><li>化学ライブラリー内の小分子のデータベースからヒット化合物を識別する。 </li></ol>

<ol>
	<li>Bayliss, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modulation+of+receptor+signaling+inhibits+regenerative+angiogenesis+in+adult+zebrafish.">Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.</a> Nature chemical biology. 2, 265-273 (2006).</li><li>Burns, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+assay+for+small+molecules+that+modulate+zebrafish+embryonic+heart+rate.">High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.</a> Nature chemical biology. 1, 263-264 (2005).</li><li>Hao, J. <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+structure-activity+relationship+study+of+dorsomorphin+analogues+identifies+selective+VEGF+and+BMP+inhibitors.">In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors.</a> ACS chemical biology. 5, 245-253 (2010).</li><li>Hong, C. C., Peterson, Q. P., Hong, J. Y., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artery/vein+specification+is+governed+by+opposing+phosphatidylinositol-3+kinase+and+MAP+kinase/ERK+signaling.">Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling.</a> Curr Biol. 16, 1366-1372 (2006).</li><li>Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drugs+that+induce+repolarization+abnormalities+cause+bradycardia+in+zebrafish.">Drugs that induce repolarization abnormalities cause bradycardia in zebrafish.</a> Circulation. 107, 1355-1358 (2003).</li><li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development.">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12965-12969 (2000).</li><li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism.">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nature chemical biology. 4, 33-41 (2008).</li><li>Zon, L. I., Peterson, R. T. <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+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature reviews. 4, 35-44 (2005).</li><li>Stern, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecules+that+delay+S+phase+suppress+a+zebrafish+bmyb+mutant.">Small molecules that delay S phase suppress a zebrafish bmyb mutant.</a> Nature chemical biology. 1, 366-370 (2005).</li><li>Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+suppression+of+a+genetic+mutation+in+a+zebrafish+model+of+aortic+coarctation.">Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation.</a> Nature biotechnology. 22, 595-599 (2004).</li></ol>

利害の衝突は宣言されません。

ゼブラフィッシュベースの化学スクリーンを計画する場合は、特に注意が検討中の表現型とそのような表現型のバックグラウンドレートの堅牢性に注意する必要があります。これは、誘導表現型の化学的抑制のためのスクリーンのために特に重要です。例えば、導入遺伝子の熱ショック誘導による表現型のため、表現型を誘導する条件は、再現性良く正確に容認できないほど高い偽陽性率を?...

<ol><li>目的の遺伝子型の大人のゼブラフィッシュの20組の最小値。 </li><li>取り外し可能なインナーコンテナと仕切り（水生生息地）と魚網、繁殖タンク、。 </li><li>ペトリ皿（10センチ）。 </li><li>プラスチック製の茶こし。 </li><li>ボトルcontaing胚の水を洗浄してください。 </li><li>使い捨てポリエチレン転送ピペット。 </li><li>ポリスチレン96穴丸底アッセイプレート（コーニングCOSTAR、ローウェル、マサチューセッツ州）。 </li><li>ガラスパスツールピペット（フィッシャーサイエンティフィック）。 </li><li>手動ピペットポンプ、10 mLの（ベルアート製品、ペクアノック、NJ）。 </li><li> E3胚の培地：0.003％PTUを（フェニルチオ-カルバミド、シグマ、セントルイス、MO）を含有する5 mMのNaCl、0.17 mMの塩化カリウム、0.33 mMのCaCl 2を 、0.33 mMのMgSO 4を 、。 PTUは、E3胚の培地1 Lに0.3 - gオプションPTUを溶解することにより10倍の溶液として調製することができます。 PTUを含む溶液をアルミホイルで覆うことにより、光から保護する必要があります。 </li><li> 12チャンネルピペット、20から20μL（エッペンドルフ）。 </li><li> 12チャンネルピペット、300〜300μL（エッペンドルフ）。 </li><li>使い捨てポリスチレンピペット流域、50 mLの（フィッシャーサイエンティフィック）。 </li><li> DMSO中の10mMストックで96 - WLL形式に配列された構造的に多様な化合物の小分子ライブラリ。各マスタープレートは96ウェルポリプロピレンストレージプレート（コーニング）に分注し、-80℃で使用時まで保存されます。 </li><li> 96ウェルプレート（Nunc、ロチェスター、ニューヨーク州）のためのアルミシールテープ。 </li><li> DMSO（シグマ、セントルイス、MO）。 </li><li>基本的なインキュベーター、28.5 ° C（フィッシャーサイエンティフィック）。 </li><li>透過光のベース（ライカマイクロシステムズ、バノックバーン、イリノイ州）と実体顕微鏡。 </li></ol>

large scale zebrafish-based in vivo small molecule screen

彼らの小さな胚の大​​きさ、急速な発展、透明性、繁殖力、そして哺乳類に多数の分子、形態学的および生理学的類似性を考えると、ゼブラフィッシュは、強力なとして浮上している<em&gt; in vivoで</em表現ベースの薬剤の画面と化学遺伝学的解析のための&gt;プラットフォーム。ここで、我々は、ゼブラフィッシュ胚を用いた小分子の大規模スクリーニングのためのシンプルで実用的な方法を示しています。

Watch this Scientific Journal Video about Large Scale Zebrafish-Based In vivo Small Molecule Screen at JoVE.com

Large Scale Zebrafish-Based In vivo Small Molecule Screen

ゼブラフィッシュは、強力なとして浮上している<em&gt; in vivoで</em表現ベースの薬剤の画面と化学遺伝学的解析のための&gt;プラットフォーム。ここで、我々は、ゼブラフィッシュ胚を用いた小分子の大規模スクリーニングのためのシンプルで実用的な方法を示しています。

大規模ゼブラフィッシュベース in vivoで小分子スクリーン

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

large-scale-zebrafish-based-in-vivo-small-molecule-screen

Research

JoVE Journal

Biology

Large Scale Zebrafish-Based In vivo Small Molecule Screen

13.4K ビュー.  Vanderbilt University School of Medicine. ゼブラフィッシュは、強力なとして浮上している in vivoでプラットフォーム。ここで、我々は、ゼブラフィッシュ胚を用いた小分子の大規模スクリーニングのためのシンプルで実用的な方法を示しています。

ビデオ: Large Scale Zebrafish-Based In vivo Small Molecule Screen

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

メタゲノムライブラリの大規模スクリーン

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

生物学

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

全モンキー脳の大規模なタンパク質の検出のためのバッチの免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

分離と成人ヒト内皮コロニー形成前駆細胞の大規模拡張

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

小分子の誘導と多能性ヒト胚性幹細胞からヒト心筋前駆体と心筋細胞の効率的な導出

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Small-scale Nuclear Extracts for Functional Assays of Gene-expression Machineries

A great deal of progress in understanding gene expression has been made using in vitro systems. For most studies, functional assays are carried out using extracts that are prepared in bulk from 10-50 or more liters of cells grown in suspension. However, these large-scale preparations are not amenable to rapidly testing in vitro effects that result from a variety of in vivo cellular treatments or conditions. This journal video article shows a method for preparing functional small-scale nuclear extracts, using HeLa cells as an example. This method is carried out using as few as three 150 mm plates of cells grown as adherent monolayers. To illustrate the efficiency of the small-scale extracts, we show that they are as active as bulk nuclear extracts for coupled RNA Polymerase II transcription/splicing reactions. To demonstrate the utility of the extract protocol, we show that splicing is abolished in extracts prepared from HeLa cells treated with the splicing inhibitor drug E7107. The small-scale protocol should be generally applicable to any process or cell type that can be investigated in vitro using cellular extracts. These include patient cells that are only available in limited quantities or cells exposed to numerous agents such as drugs, DNA damaging agents, RNAi, or transfection, which require the use of small cell populations. In addition, small amounts of freshly grown cells are convenient and/or required for some applications.

A protocol for preparation of robust, small-scale HeLa nuclear extracts is described. This protocol is valuable for assays that require use of small populations of cells, such as cells treated with drugs or RNAi. The method should be applicable to a wide variety of gene expression assays and other cell types, including patient cells.

遺伝子発現用機械の機能的アッセイのための小規模の核抽出物

A great deal of progress in understanding gene expression has been made using in vitro systems.  For most studies, functional assays are carried out using ...

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

大規模なSNPジェノタイピングアプリケーション用インフィニウムアッセイ

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

敏感な、大規模定量的メタボロームのための戦略

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

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 ...

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water deficit or pathogens plays a crucial role. Identifying small molecules that regulate stomatal movement can therefore contribute to understanding the physiological basis by which plants adapt to their environment. Large-scale screening approaches that have been used to identify regulators of stomatal movement have potential limitations: some rely heavily on the abscisic acid (ABA) hormone signaling pathway, therefore excluding ABA-independent mechanisms, while others rely on the observation of indirect, long-term physiological effects such as plant growth and development. The screening method presented here allows the large-scale treatment of plants with a library of chemicals coupled with a direct quantification of their transpiration by thermal imaging. Since evaporation of water through transpiration results in leaf surface cooling, thermal imaging provides a non-invasive approach to investigate changes in stomatal conductance over time. In this protocol, Helianthus annuus seedlings are grown hydroponically and then treated by root feeding, in which the primary root is cut and dipped into the chemical being tested. Thermal imaging followed by statistical analysis of cotyledonary temperature changes over time allows for the identification of bioactive molecules modulating stomatal aperture. Our proof-of-concept experiments demonstrate that a chemical can be carried from the cut root to the cotyledon of the sunflower seedling within 10 minutes. In addition, when plants are treated with ABA as a positive control, an increase in leaf surface temperature can be detected within minutes. Our method thus allows the efficient and rapid identification of novel molecules regulating stomatal aperture.

Identification of Novel Regulators of Plant Transpiration by Large-Scale Thermal Imaging Screening in Helianthus Annuus

We provide a method for identifying modulators of foliar transpiration by large-scale screening of a compound library.

ヘリアンサス・アンヌウスにおける大規模な熱画像スクリーニングによる植物蒸散の新規レギュレーターの同定

Plant adaptation to biotic and abiotic stresses is governed by a variety of factors, among which the regulation of stomatal aperture in response to water ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

大規模エピジェネティック研究のための半自動化されたChIP-Seq法

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...