MLはNIA（R00AG033176とR01AG040081）と研究所監督研究開発、エネルギー契約＃DE-AC02-05CH11231の米国防総省でサポートされています。

<ol>
	<li>Bissell, M. J., Labarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Context,+tissue+plasticity,+and+cancer:+are+tumor+stem+cells+also+regulated+by+the+microenvironment.">Context, tissue plasticity, and cancer: are tumor stem cells also regulated by the microenvironment.</a> Cancer Cell. 7, 17-23 (2005).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>McBeath, R., Pirone, D. M., Nelson, C. M., Bhadriraju, K., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+shape,+cytoskeletal+tension,+and+RhoA+regulate+stem+cell+lineage+commitment.">Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment.</a> Dev. Cell. 6, 483-495 (2004).</li><li>LaBarge, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mammary+progenitor+cell+fate+decsions+are+products+of+interactions+with+combinatorial+microenvironments.">Human mammary progenitor cell fate decsions are products of interactions with combinatorial microenvironments.</a> Integrative Biology. 1, 70-79 (2009).</li><li>Kim, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+Methods+for+Polymers+in+Cell+and+Tissue+Engineering.">Patterning Methods for Polymers in Cell and Tissue Engineering.</a> Annals of biomedical engineering. , (2012).</li><li>Boudou, T., Ohayon, J., Picart, C., Pettigrew, R. I., Tracqui, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+elastic+properties+of+polyacrylamide+gels:+implications+for+quantification+of+cellular+forces.">Nonlinear elastic properties of polyacrylamide gels: implications for quantification of cellular forces.</a> Biorheology. 46, 191-205 (2009).</li><li>Tse, J. R., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+hydrogel+substrates+with+tunable+mechanical+properties.">Preparation of hydrogel substrates with tunable mechanical properties.</a> Current protocols in cell biology. Chapter 10, Unit 10 (2010).</li><li>Flaim, C. J., Chien, S., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extracellular+matrix+microarray+for+probing+cellular+differentiation.">An extracellular matrix microarray for probing cellular differentiation.</a> Nat. Methods. 2, 119-125 (2005).</li><li>Soen, Y., Mori, A., Palmer, T. D., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+regulation+of+human+neural+precursor+cell+differentiation+using+arrays+of+signaling+microenvironments.">Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.</a> Mol. Syst. Biol. 2, 37 (2006).</li></ol>

特別な利害関係は宣言されません。

ここに提示されMEArrayの方法は、細胞と組み合わせ微小環境との相互作用4の機能解析を可能にします。 MEArray分析は、基本的な微細化技術、細胞生物学、および多くのマルチ施設で利用可能なマイクロアレイプリンティングロボットや分析装置の使用を兼ね備えています。無血清培地製剤は添付ファイルを向上させることができますBSAまたは&lt;1％の血清を含むようにいくつかのケー?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td>ガラスは、25ミリメートル×75ミリメートルをスライド</td><td> VWR </td><td> 48311-600 </td><td></td></tr><tr><td>ガラスカバースリップ（その1）24ミリメートル×50ミリメートル</td><td> VWR </td><td> 48393-241 </td><td></td></tr><tr><td>染色皿（またはCOPLAN瓶） </td><td> VWR </td><td> 25461-003 </td><td></td></tr><tr><td>ペトリ皿（15センチ） </td><td> BDファルコン</td><td> 351058 </td><td></td></tr><tr><td>のNaOH（1.0N） </td><td>シグマアルドリッチ</td><td> S2567 </td><td></td></tr><tr><td> APES（&gt; 98％（3 - アミノプロピル）トリエトキシシラン） </td><td>シグマアルドリッチ</td><td> A3648 </td><td></td></tr><tr><td>グルタルアルデヒド</td><td>シグマアルドリッチ</td><td> G7651 </td><td>水中の50％ </td></tr><tr><td> APS（&gt; 98％過硫酸アンモニウム） </td><td> Sigmaアルドリッチ</td><td> A3678 </td><td> ddH 2 Oで10パーセントの作業溶液を調製</td></tr><tr><td> TEMED（N、N、N &#39;、N&#39;-テトラメチルエチレンジアミン） </td><td>シグマアルドリッチ</td><td> T9281 </td><td></td></tr><tr><td>アクリルアミド（40％） </td><td>シグマアルドリッチ</td><td> A4058 </td><td></td></tr><tr><td>ビスアクリルアミド（2％w / v）の</td><td>フィッシャーBioReagents </td><td> BP1404-250 </td><td></td></tr><tr><td> 0.45μmのシリンジフィルター4mmのナイロン</td><td>ナルゲン</td><td> 176-0045 </td><td></td></tr><tr><td> FITC </td><td>シグマアルドリッチ</td><td> F4274 </td><td></td></tr><tr><td> PDMS（ポリジメチルシロキサン） </td><td>ダウコーニング</td><td> 3097358-1004 </td><td>エルズワース接着剤を介して、シルガード184エラストマーキット</td></tr><tr><td> 2チャンバースライド</td><td> NUNC </td><td> 177380 </td><td></td></tr><tr><td>プルロニックF108 </td><td> BASF </td><td> 30089186 </td><td></td></tr><tr><td> AQUariumシーラント</td><td>ダウコーニング</td><td> DAP 00688 </td><td></td></tr><tr><td> Fluormount-G </td><td>サザンバイオテクノロジー</td><td> 0100から01 </td><td></td></tr><tr><td>使い捨てのプラスチック製コップ</td><td></td><td></td><td></td></tr><tr><td>舌圧子</td><td></td><td></td><td></td></tr><tr><td>ニトリル手袋</td><td></td><td></td><td></td></tr><tr><td>プラスチック製の顕微鏡スライドボックス</td><td></td><td></td><td></td></tr><tr><td>スピンコーター</td><td> WS-400B-6NPP/LITE </td><td> Laurellテクノロジーズ株式会社</td><td></td></tr><tr><td>オーブン</td><td></td><td></td><td></td></tr><tr><td>デジタルホットプレート</td><td></td><td></td><td></td></tr><tr><td> 384ウェルプレート</td><td></td><td></td><td>マイクロアレイロボットに適したブランド</td></tr><tr><td>マイクロアレイプリンティングロボット</td><td></td><td></td><td></td></tr><tr><td>逆位相と蛍光顕微鏡</td><td></td><td></td><td></td></tr><tr><td>軸索マイクロアレイスキャナー</td><td>分子デバイス</td><td></td><td>複数のコンフィギュレーションが存在し</td></tr></tbody></table>

fabrication and use of microenvironment microarrays (mearrays)

細胞とその周囲の微小環境との相互作用が細胞挙動のために機能的な結果をもたらす。単一細胞レベルでは、異なる微小環境は、分化、遊走および増殖表現型を課すことができ、組織レベルでの微小環境は、形態形成および腫瘍形成1のような複雑な処理します。のみならず、内微小環境への影響の細胞および分子細胞内容物をしますが、とても弾力性2、組織の幾何学3を行う。細胞間、ECM-、および-可溶性因子の相互作用の総和として定義されて、物理的な特性に加えて、微小環境は複雑である。組織内の細胞の表現型は、それらのゲノムコンテンツやmicroenviroment持つ組み合わせの相互作用に起因して部分的に部分的に起因している。主要な課題は、独特の行動と微小環境の成分の特定の組み合わせをリンクすることです。 ENT &quot;&gt;ここでは、コンビナトリアル4微小環境との相互作用の細胞ベースの機能的スクリーニングのための微小環境マイクロアレイ（MEArray）プラットフォームを提示します。方法は分子組成と弾性率の同時制御を可能にし、広く利用可能なマイクロアレイの使用を組み合わせと微細技術。MEArray画面では、以下のよう成体前駆細胞などの珍しい種類の細胞の機能研究を容易にアレイあたり10,000細胞、できるだけ少ない必要があります。テクノロジーの限界は、その全体組織微小環境が完全MEArraysに要約されることができないですが、との比較多数の関連微小環境に同じ細胞型の応答は、例えば特定の組織を特徴付けるECMタンパク質のペアの組み合わせは、微小環境の成分が組織特異的機能の表現型を引き出す方法についての洞察を提供します。 MEArraysは、組換えGROの多種多様を使用して印刷することができますwthの因子、サイトカイン、および精製されたECMタンパク質、およびそれらの組み合わせが含まれる。プラットフォームは、特定の試薬の利用可能性によって制限されています。 MEArraysは、時間経過の分析に適しているが、ほとんどの場合、蛍光プローブを用いた測定可能である細胞の機能のエンドポイントの解析に使用されています。例えば、DNA合成、アポトーシス、分化した状態の取得、または特定の遺伝子産物の生産は、一般的に測定されています。簡単に言えば、MEArray実験の基本的な流れは、印刷基層でコーティングスライドを準備し、印刷されるタンパク質のマスタープレートを準備することです。アレイはマイクロアレイロボットで印刷されその後、細胞培養中で成長し、接続を許可され、その後、化学実験のエンドポイントに達した時点で固定されています。従来の顕微鏡やマイクロアレイスキャナーで画像化蛍光または比色アッセイは、関連する分子や細胞の表現型を示す（ 図1）を明らかにするために使用されています。

Watch this Scientific Journal Video about Fabrication and Use of MicroEnvironment microArrays MEArrays at JoVE.com

Fabrication and Use of MicroEnvironment microArrays MEArrays

細胞機能上の微小環境の分子組成の影響についての洞察を得るためのコンビナトリアル機能的スクリーニング方法が記載されている。この方法は、細胞接着および機能解析をサポートしていることを定義したコンビナトリアル微小環境の配列を生成するために、既存のマイクロアレイベースの技術を活用しています。

微小環境マイクロアレイの作製と利用（MEArrays）

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

fabrication-and-use-of-microenvironment-microarrays-mearrays

Research

JoVE Journal

Biology

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

9.9K ビュー.  Lawrence Berkeley National Laboratory. 細胞機能上の微小環境の分子組成の影響についての洞察を得るためのコンビナトリアル機能的スクリーニング方法が記載されている。この方法は、細胞接着および機能解析をサポートしていることを定義したコンビナトリアル微小環境の配列を生成するために、既存のマイクロアレイベースの技術を活用しています。

ビデオ: Fabrication and Use of MicroEnvironment microArrays MEArrays

Bacterial Gene Expression Analysis Using Microarrays

マイクロアレイを用いた細菌の遺伝子発現解析

生物学

PDMS Device Fabrication and Surface Modification

PDMSデバイスの作製と表面改質

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

アンペロメトリー電極の作製

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

筋原培養組織コンストラクトの作製

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Genome-wide Analysis of Aminoacylation (Charging) Levels of tRNA Using Microarrays

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both prokaryotic and eukaryotic cells lead to translational regulation which is a major cellular mechanism of stress response. Familiar examples are the stringent response in E. coli and the Gcn2 stress response pathway in yeast ([2-6]). Recent work in E. coli and S. cerevisiae have shown that tRNA charging patterns are highly dynamic and depends on the type of stress experienced by cells [1, 6, 7]. The highly dynamic, variable nature of tRNA charging makes it essential to determine changes in tRNA charging levels at the genomic scale, in order to fully elucidate cellular response to environmental variations. In this review we present a method for simultaneously measuring the relative charging levels of all tRNAs in S. cerevisiae . While the protocol presented here is for yeast, this protocol has been successfully applied for determining relative charging levels in a wide variety of organisms including E. coli and human cell cultures[7, 8].

Genome-wide Analysis of Aminoacylation Charging Levels of tRNA Using Microarrays

We describe a method for microarray analysis to determine relative aminoacylation levels of all tRNAs from S. cerivisiae.

マイクロアレイを用いたtRNAのアミノアシル化（充電）レベルのゲノムワイド解析

tRNA aminoacylation, or charging, levels can rapidly change within a cell in response to the environment[1]. Changes in tRNA charging levels in both ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNAマイクロアレイ：サンプルの品質管理、アレイハイブリダイゼーションとスキャニング

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

ヒトタンパ​​ク質アトラス内の組織マイクロアレイ、免疫組織化学染色とデジタル化の生産

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

マイクロアレイを用いた植物細胞壁ポリマーの糖鎖プロファイリング

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression3-6 and other characteristics of cultures at steady-state equilibrium.7 Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.8 A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. 
We present here the design and operation of a relatively simple, low cost array of miniature chemostats&mdash;or ministats&mdash;and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.

We developed and validated a small-footprint array of miniature chemostats built from readily available parts for low cost. Physiological and experimental evolution results were similar to larger volume chemostats. The ministat array provides a compact, inexpensive, and accessible platform for traditional chemostat experiments, functional genomics, and chemical screening applications.

多重ケモスタットアレイの設計および使用

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

タンパク質 - タンパク質相互作用を検出するために、高密度機能タンパク質マイクロアレイのプロービング

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Methods Article

微小環境マイクロアレイの作製と利用（MEArrays）