我々は、沿岸と外洋水の低酸素領域で進行中の研究を支援するためのイノベーション、ブリティッシュコロンビア州の知識開発基金と国立科学およびカナダの工学研究審議会（NSERC）のためのカナダの財団に感謝の意を表します。 MTとSLは、微生物の多様性と進化（CMDE）用トゥーラ基礎資金センターからの奨学金によってサポートされていました。 MTはまた、ドイツ学術振興協会（DFG）から奨学金の支援を受けた。

<ol>
	<li>Zaikova, E., Hawley, A., Walsh, D. A., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seawater+sampling+and.">Seawater sampling and.</a> J Vis Exp. , (2009).</li><li>Walsh, D. A., Zaikova, E., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+volume+(20L+)+filtration+of+coastal+seawater+samples.">Large volume (20L+) filtration of coastal seawater samples.</a> J Vis Exp. 28, (2009).</li><li>Wright, J. J., Lee, S., Zaikova, E., Walsh, D. A., Hallam, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Extraction+from+0.22+&#956;M+Sterivex+Filters+and+Cesium+Chloride+Density+Gradient+Centrifugation.">DNA Extraction from 0.22 &#956;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation.</a> J Vis Exp. 31, 10-3791 (2009).</li><li>Stein, J. L., Marsh, T. L., Wu, K. Y., Shizuya, H., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+uncultivated+prokaryotes:+isolation+and+analysis+of+a+40-kilobase-pair+genome+fragment+from+a+planktonic+marine+archaeon.">Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon.</a> J. Bacteriol. 178, 591-599 (1996).</li><li>B&#233;j&#224;, O., Suzuki, O., Koonin, M. T., Aravind, E. V., Hadd, L., Nguyen, L. P. A., Villacorta, R., Amjadi, M., Garrigues, C., Jovanovich, S. B., Feldman, R. A., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+analysis+of+bacterial+artificial+chromosome+libraries+from+a+marine+microbial+assemblage.">Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage.</a> Environ. Microbiol. 2, 516-529 (2000).</li><li>DeLong, E. F., Preston, C. M., Mincer, T., Rich, V., Hallam, S. J., Frigaard, N. -. U., Martinez, A., Sullivan, M. B., Edwards, R., Brito, B. R., Chisholm, S. W., Karl, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Community+genomics+among+stratified+microbial+assemblages+in+the+Ocean's+interior.">Community genomics among stratified microbial assemblages in the Ocean's interior.</a> Science. 311, 496-503 (2006).</li><li>Hallam, S. J., Putnam, N., Preston, C. M., Detter, J. C., Rokhsar, D., Richardson, P. M., DeLong, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+methanogenesis:+testing+the+hypothesis+with+environmental+genomics.">Reverse methanogenesis: testing the hypothesis with environmental genomics.</a> Science. 305, 1457-1462 (2004).</li></ol>

手順は、最も効率的に沿岸水のサンプルに由来するゲノムDNAを持つ大規模挿入フォスミドライブラリを生成する方法を説明しています。アップストリームゲノムDNAの抽出は、別々のプロトコル[3]に記述されています。 フォスミドライブラリとしての生産は、すべての4つ表示される手順を含む、全体の手続きに間に合うように多段階のプロセス、計画は少なくとも2〜3週?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Step II</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CopyControl&trade; Fosmid Library Production Kit</td>
<td>&nbsp;</td>
<td>EPICENTRE</td>
<td>CCFOS110</td>
<td>sufficient to generate 9-10 fosmid libraries</td>
</tr>
<tr>
<td>SeaPlaque Agarose</td>
<td>&nbsp;</td>
<td>Cambrex</td>
<td>50100</td>
<td>&nbsp;</td>
<tr>
<td>stirring hot plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>PC-420D</td>
<td>&nbsp;</td>
<tr>
<td>1.0X TAE running buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CHEF Mapper XA pulsed field electrophoresis system including cooling module and variable speed pump</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>&lambda; DNA-HindIII Digest</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>N3012S</td>
<td>&nbsp;</td>
<tr>
<td>MidRange I PFG Marker</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>N3551S</td>
<td>&nbsp;</td>
<tr>
<td>10,000X SYBR Gold</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S11494</td>
<td>&nbsp;</td>
<tr>
<td>microwavable plastic wrap</td>
<td>&nbsp;</td>
<td>Saran premium wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Safe Imager transilluminator</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S37102</td>
<td>&nbsp;</td>
<tr>
<td>GELase Agarose Gel-Digesting Preparation</td>
<td>&nbsp;</td>
<td>EPICENTRE</td>
<td>G09100</td>
<td>&nbsp;</td>
<tr>
<td>scalpel</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-927-5D</td>
<td>&nbsp;</td>
<tr>
<td>Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>UFC801024</td>
<td>&nbsp;</td>
<tr>
<td>Microcon YM-30 Centrifugal Filter Unit </td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>42410</td>
<td>&nbsp;</td>
<tr>
<td>nuclease free water</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM9932</td>
<td>&nbsp;</td>
<tr>
<td>Quant-iT PicoGreen dsDNA Assay Kit *2000 assays*#</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P7589</td>
<td>&nbsp;</td>
<tr>
<td>digital dry block heater</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>12621-088</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>14231-854</td>
<td>&nbsp;</td>
<tr>
<td>centrifuge</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>Avanti J-E</td>
<td>&nbsp;</td>
<tr>
<td>centrifuge rotor</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>JA 5.3</td>
<td>&nbsp;</td>
<tr>
<td>table top centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>5415D</td>
<td>&nbsp;</td>
<tr>
<td>microcentrifuge tubes, 1.7 mL, clear</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT-175-C</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes </td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>430052</td>
<td>&nbsp;</td>
<tr>
<td>Step III</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP 1426-2</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>M2643</td>
<td>&nbsp;</td>
<tr>
<td>phage dilution buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris-HCl [pH 8.3], 100 mM NaCl, 10 mM MgCl2</td>
</tr>
<tr>
<td>cryogenic vials</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>430488</td>
<td>&nbsp;</td>
<tr>
<td>Step IV</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>96 well plate with lid</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>CS003370(3370)</td>
<td>&nbsp;</td>
<tr>
<td>384 well plate with lid</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>07201157(3680)</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 100 O.D. x 15mm H</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes 150 Dia. x 15mmH</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-14</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon BioDish XL 245 X 245 mm</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CABD351040</td>
<td>&nbsp;</td>
<tr>
<td>glycerol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G5516</td>
<td>&nbsp;</td>
<tr>
<td>chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C0378</td>
<td>&nbsp;</td>
<tr>
<td>LB broth</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP 1426-2</td>
<td>&nbsp;</td>
<tr>
<td>Difco Agar</td>
<td>&nbsp;</td>
<td>BD</td>
<td>214530</td>
<td>&nbsp;</td>
<tr>
<td>glass beads</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>10-310-1</td>
<td>&nbsp;</td>
<tr>
<td>QFill3</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>X3050</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>Javex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E7023</td>
<td>&nbsp;</td>
<tr>
<td>15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Fisher-Corning</td>
<td>430052</td>
<td>&nbsp;</td>
<tr>
<td>QPix colony picker</td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>QPix2</td>
<td>&nbsp;</td>
<tr>
<td>picking head (E. coli) </td>
<td>&nbsp;</td>
<td>Genetix</td>
<td>X4006</td>
<td>&nbsp;</td>		</tbody>
		</table>
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large insert environmental genomic library production

自然界における微生物の大半は現在、従来の栽培方法にアクセスできないままです。過去10年間、カルチャに依存しない環境ゲノム（<em&gt;すなわち</em&gt;メタゲノム）アプローチは、研究者は環境から直接土着微生物群集の遺伝的内容をキャプチャすることで、この栽培のギャップを埋めることが可能に、浮上している。この目的のために、ゲノムDNAライブラリーは、巧みな実験室のクローニング技術とはいえ、標準を使用して構築されています。ここでは、季節的低酸素フィヨルドの垂直方向の深さの連続体に由来するDNAで大規模な挿入、環境ゲノムフォスミドライブラリーの構築について説明します。このプロトコルは直接沿岸海洋の水の採取[1]などの接続プロトコルのシリーズにリンクされている微生物バイオマスの、大量のろ過[2]とDNA抽出と浄化のプロトコル[3]。当初は、高品質のゲノムDNAは、5 - リン酸化平滑末端の作成を終了 - 修理です。エンド修理DNAは、サイズの選択とゲル抽出を30と6万塩基対（KB）の長さの間のDNA断片を回収するために行われるため、パルスフィールドゲル電気泳動（PFGE）を施す。サイズ選択されたDNAをPFGEゲルマトリックスから精製し、ホスファターゼ処理、平滑末端フォスミドCopyControlベクトルPCC1（EPICENTREに連結され<a href="http://www.epibio.com/item.asp?ID=385"&gt; http://www.epibio.com/item.asp?ID=385</a&gt;）。 PCC1とインサートDNAのリニアコンカテマーは、その後、ファージ耐性のその後の感染症で、ラムダterminaseによってファージ粒子にパッケージheadfullされています<em&gt; E.大腸菌</em&gt;細胞。成功した形質導入クローンを抗生物質選択の下にLB寒天プレートに回収し、ロボットがピッキング、自動コロニー（Qpix2、GENETIX）を用いて384ウェルプレートフォーマットでアーカイブされます。現在のプロトコルは、（4）〜（7）をEPICENTREからCopyControlフォスミドライブラリーの制作キットを含むさまざまなソースから描画し、複数の研究グループの出版された作品。各ステップは心のベストプラクティスが提示されます。可能な限り我々はライブラリの生産の全体的な品質と効率を向上させるために実行に微妙な点を強調。フォスミドライブラリーの製造と自動コロニーピッキングのプロセス全体が含まれている多くのインキュベーションのステップがあるとして、少なくとも7〜10日かかる。しかし、プロトコル内で言及されている可能性がいくつかの停止ポイントがあります。

Watch this Scientific Journal Video about Large Insert Environmental Genomic Library Production at JoVE.com

Large Insert Environmental Genomic Library Production

季節低酸素フィヨルドの垂直方向の深さの連続体から分離された環境ゲノムDNAとのフォスミドライブラリーの構築が記載されている。得られたクローンのライブラリーは384ウェルプレートに取り、自動コロニーピッキングシステムのアプリケーションによって下流の塩基配列決定と機能的スクリーニングのためにアーカイブされます。

大規模な挿入環境ゲノムライブラリーの製造

The vast majority of microbes in nature currently remain inaccessible to traditional cultivation methods. Over the past decade, culture-independent ...

large-insert-environmental-genomic-library-production

Research

JoVE Journal

Biology

15.8K ビュー.  University of British Columbia - UBC. 季節低酸素フィヨルドの垂直方向の深さの連続体から分離された環境ゲノムDNAとのフォスミドライブラリーの構築が記載されている。得られたクローンのライブラリーは384ウェルプレートに取り、自動コロニーピッキングシステムのアプリケーションによって下流の塩基配列決定と機能的スクリーニングのためにアーカイブされます。

ビデオ: Large Insert Environmental Genomic Library Production

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

Isolation of Genomic DNA from Mouse Tails

マウスの尾からのゲノムDNAの単離

Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.

This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.

全ゲノムアレイ比較ゲノムハイブリダイゼーションの技術デモンストレーション

Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1.  Recent ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

接着細胞培養用の酸素挿入の作製と動作

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Genomic MRI - a Public Resource for Studying Sequence Patterns within Genomic DNA

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in their nucleotide composition and consist of a complex mosaic of sequence patterns. These patterns include so-called Mid-Range Inhomogeneity (MRI) regions -- sequences 30-10000 nucleotides in length that are enriched by a particular base or combination of bases (e.g. (G+T)-rich, purine-rich, etc.). MRI regions are associated with unusual (non-B-form) DNA structures that are often involved in regulation of gene expression, recombination, and other genetic processes (Fedorova &amp; Fedorov 2010). The existence of a strong fixation bias within MRI regions against mutations that tend to reduce their sequence inhomogeneity additionally supports the functionality and importance of these genomic sequences (Prakash et al. 2009).
Here we demonstrate a freely available Internet resource -- the Genomic MRI program package -- designed for computational analysis of genomic sequences in order to find and characterize various MRI patterns within them (Bechtel et al. 2008). This package also allows generation of randomized sequences with various properties and level of correspondence to the natural input DNA sequences. The main goal of this resource is to facilitate examination of vast regions of non-coding DNA that are still scarcely investigated and await thorough exploration and recognition.

We present a public computational web site for the analysis of genomic sequences. It detects DNA sequence patterns with various non-random nucleotide compositions. This resource also generates randomized sequences with diverse levels of complexity.

ゲノムMRI - ゲノムDNA内の配列パターンを研究するためのパブリックリソース

Non-coding genomic regions in complex eukaryotes, including intergenic areas, introns, and untranslated segments of exons, are profoundly non-random in ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

バーコード酵母ライブラリーの競争力のあるゲノム画面

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryoteのゲノム形質転換<em&gt; Ostreococcusタウリ</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

ゲノムコピー数変異体の検出のためのアレイ比較ゲノムハイブリダイゼーション（アレイCGH）

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

A Two-Step Strategy that Combines Epigenetic Modification and Biomechanical Cues to Generate Mammalian Pluripotent Cells

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe a new strategy that combines the use of chemical epigenetic erasing with mechanosensing-related cues, to generate mammalian pluripotent cells. Two main steps are required. In the first step, adult mature (terminally differentiated) cells are exposed to the epigenetic eraser 5-aza-cytidine to drive them into a pluripotent state. This part of the protocol was developed, based on the increasing understanding of the epigenetic mechanisms controlling cell fate and differentiation, and involves the use of the epigenetic modifier to erase cell differentiated state and then drive into a transient high plasticity window.
In the second step, erased cells are encapsulated in polytetrafluoroethylene (PTFE) micro-bioreactors, also known as Liquid Marbles, to promote 3D cell rearrangement to extend and stably maintain the acquired high plasticity. PTFE is a non-reactive hydrophobic synthetic compound and its use permits the creation of a cellular microenvironment, which cannot be achieved in traditional 2D culture systems. This system encourages and boosts the maintenance of pluripotency though bio-mechanosensing-related cues.
The technical procedures described here are simple strategies to allow for the induction and maintenance of a high plasticity state in adult somatic cells. The protocol allowed the derivation of high plasticity cells in all mammalian species tested. Since it does not involve the use of gene transfection and is free of viral vectors, it may represent a notable technological advance for translational medicine applications. Furthermore, the micro-bioreactor system provides a notable advancement in stem cell organoid technology by in vitro re-creating a specific micro-environment that allows for the long-term culture of high plasticity cells, namely as ESCs, iPSCs, epigenetically erased cells and MSCs.

We here present a method that combines the use of chemical epigenetic erasing with mechanosensing-related cues to efficiently generate mammalian pluripotent cells, without the need of gene transfection or retroviral vectors. This strategy is, therefore, promising for translational medicine and represents a notable advancement in stem cell organoid technology.

エピジェネティック修飾と生体力学的手がかりを組み合わせて哺乳類多能性細胞を生成する2段階戦略

Cell phenotype can be reversed or modified with different methods, with advantages and limitations that are specific for each technique. Here we describe ...

A Robust Method for the Large-Scale Production of Spheroids for High-Content Screening and Analysis Applications

High-content screening (HCS) and high-content analysis (HCA) are technologies that provide researchers with the ability to extract large-scale quantitative phenotypic measurements from cells. This approach has proved powerful for deepening our understanding of a wide range of both fundamental and applied events in cell biology. To date, the majority of applications for this technology have relied on the use of cells grown in monolayers, although it is increasingly realized that such models do not recapitulate many of the interactions and processes that occur in tissues. As such, there has been an emergence in the development and use of 3-dimensional (3D) cell assemblies, such as spheroids and organoids. Although these 3D models are particularly powerful in the context of cancer biology and drug delivery studies, their production and analysis in a reproducible manner suitable for HCS and HCA present a number of challenges. The protocol detailed here describes a method for the generation of multicellular tumor spheroids (MCTS), and demonstrates that it can be applied to three different cell lines in a manner that is compatible with HCS and HCA. The method facilitates the production of several hundred spheroids per well, providing the specific advantage that when used in a screening regime, data can be obtained from several hundred structures per well, all treated in an identical manner. Examples are also provided, which detail how to process the spheroids for high-resolution fluorescence imaging and how HCA can extract quantitative features at both the spheroid level as well as from individual cells within each spheroid. This protocol could easily be applied to answer a wide range of important questions in cell biology.

This protocol details a method for the production of three different types of spheroids in a manner that makes them suitable for large-scale high-content screening and analysis. In addition, examples are presented showing how they can be analyzed at spheroid and individual cell levels.