We thank Stephen Blazie, Karen Anderson, Josh LaBaer for advice and discussion. Karen Anderson, John Chaput, and Josh LaBaer for sharing reagents and instrumentation, Michael Gaskin and Andrea Throop for technical advise and protocols. Justin Wolter is a Maher scholar and thanks the Maher family for their generous support.

<ol>
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This work is supported by funds from the College of Liberal Arts and Science and the Biodesign Institute at Arizona State University and NIH Exploratory/Developmental Research Grant 1R21CA179144-01A1.

3&#39;LIFEアッセイは、高スループットでの3&#39;UTR中の機能のmiRNAのターゲットを識別します。このアッセイは、実験的に興味のあるmiRNAのための推定上の多数のターゲットを特定したい研究者のために有用です。アッセイがmiRNAの標的の機能的尺度を提供し、単一のmiRNAに対する3&#39;UTR ::シングルレポーターのバイナリテストが自信を持って取り組むことができるという点で、3&#39;LIFEアッセイ?...

この方法の全体的な目標は、検出し、正確に高スループットでマイクロRNA（miRNAの）ターゲットをマッピングすることです。 miRNAは、長さが約22ヌクレオチドの内因性の非コードRNAです。転写および処理に続いて、成熟miRNAは、タンパク質複合体中に組み込まれる（RISC）、複雑なRNA誘導サイレンシングと呼ばれます。各miRNAは、翻訳抑制またはmRNA切断1のいずれかで得られた、主にメッセンジャーRNAの3 &#39;非翻訳領域（3&#39;UTRを）（mRNAを）に位置要素を標的とするためにRISCを誘導します。 miRNAは、標準的なワトソン - クリックしてGに基づいて、標的部位を認識する：Uは、塩基対形成をウォブルし、本質的に縮重している、複数のミスマッチ塩基対を含有し、領域を膨らみました。多くのmiRNAが広く、彼らは生物学的役割の多様な範囲を再生する人間2,3に植物から保存されています。後生動物ではmiRNAは、細胞運命の決定4、発生のタイミング5を含む複数の生物学的プロセスに影響を与えることができます</sup&gt;、および頻繁に組織特異的な発現パターン6,7を示します。誤発現のmiRNAは、標的遺伝子の機能のみに基づいて細胞の挙動に大きな影響を持つことができ、異常な遺伝子調節をもたらすことができます。このように、miRNAは、神経変性8,9、糖尿病10及び11を含む癌疾患の広範囲に連結されています。バイオインフォマティクスとウェットベンチアプローチは各miRNAは、ハイスループットまたはゲノムワイドアプローチは潜在的な相互作用のこの大きなプールをプローブするために必要であることを示している、別個のmRNA 12-14の千数百人を標的にすることが可能であり得ることを示唆しています。 標的遺伝子を同定することは機構的に定義するmiRNAの機能の重要なコンポーネントであり、そうするために研究者が大規模に目標を明らかにすることができなければなりません。いくつかのアプローチは、バイオインフォマティクスの予測アルゴリズムを含む、miRNAの標的を同定するためのハイスループットシークエンシングが開発されていますターゲットとするmRNA、およびレポーターに基づくアッセイの。これらのアプローチのそれぞれが固有の長所と短所を持っています。 miRNAのターゲティングは、配列特異性によって案内されることを考えると、最も特にmiRNAのヌクレオチド2~6の、いくつかのアルゴリズムは、多くの生物のゲノム全体のmiRNA標的を予測するために開発されてきた（シード領域と呼ばれます）。これらのアルゴリズムは、検証済みのmiRNA標的の観察された塩基対形成のモチーフを使用して訓練し、頻繁にこのような厳しいシードのペアリング、サイトの保全、および/ ​​または熱力学的安定性15などのパラメータを利用しています。これらのフィルタは唯一の信頼度の高いターゲットに十分な相補性を有する推定多数のターゲットを絞り込みながら、彼らは、最近の証拠は16-24普及している示唆している種特異的および非標準のmiRNA標的部位を、除外することができます。また、これらの予測は、このような代替ポリアデニル化などのmiRNA標的部位を除外mRNAプロセシングのための仕組み、に取ることはありません25、26 RNA 編集 、RNAのメチル化27、及び協同的結合。このように、高い偽陽性および偽陰性率は、多くのアルゴリズム22,24,28のために報告されています。これらのアルゴリズムは、その後の実験的検証のための候補miRNAの標的を同定するのに有用であるが、これらの高いエラー率が系統的miRNA標的の検出のためのバイオインフォマティクスアプローチの有効性を制限します。 体系的に与えられたmiRNAの間の相互作用および潜在的標的と3&#39;UTRを探索するために、我々はの3&#39;UTR（3&#39;LIFE）24での機能要素の蛍光同定と呼ばれるハイスループットアッセイを開発しました。このアッセイは、直接的な相互作用およびデュアルルシフェラーゼレポーターシステムを使用してクエリのmiRNAによる試験3&#39;UTRの翻訳抑制。この系において、目的の遺伝子の3&#39;UTRは、ホタルルシフェラーゼ（Fluc細胞）レポーターリーディングフレームの下流にクローニングされます。レポーター短所tructは、HEK293T細胞内のクエリのmiRNAで共トランスフェクトされます。 miRNAのターゲティングは、試験Fluc細胞:: 3&#39;UTRレポーターおよび第非特異的ウミシイタケルシフェラーゼレポーターの間の相対的な変化を測定することによって決定されます。重要なのは、ルシフェラーゼアッセイは、レポーターの翻訳出力に影響を与える機能のmiRNA / mRNAの相互作用を検出します。このことは、これは3&#39;UTR基づく規制の独立したタンパク質の存在量のmRNAの分解や翻訳抑制の違いだけでなく、変化を迂回するには、そのようなRT-qPCRのやウェスタンブロットなどのmiRNA調節を検出する従来の方法に比べて重要な利点です。 ルシフェラーゼアッセイは、広く、それらの相対的な単純性と感度の直接のmiRNAの標的を検証するために利用されている、まだハイスループットスクリーニングにおけるそれらの使用は、高い消耗品試薬に関連するコスト、公共の情報源からの3&#39;UTRライブラリーの欠如、および存在しないことによって制限されています標準化されたルシフェラーゼのprotのocols、複数のデータセット全体での機能抑制を比較する際に困難をもたらします。 3&#39;LIFEアッセイの使用を容易にするために、我々は定期的に更新して展開され3&#39;UTRライブラリを作成、実験デザイン、非商業的なトランスフェクション24、ルシフェラーゼ試薬29の利用の簡素化に重点を置いて、を介して利用できました公共プラスミドリポジトリ30。 3&#39;LIFEアッセイのスケーラビリティは、バイオインフォマティクス同定された遺伝子の方の画面にバイアスをかけることなく、与えられたmiRNAによって標的化するための大きな3&#39;UTRライブラリーのスクリーニングを可能にします。標準的な予測の相互作用を試験することに加えて、この体系的なアプローチは、非標準および/または種特異的な相互作用を介して駆動される新規標的の同定を可能にします。重要なことは、タンパク質の産生を対象miRNAの効果は、一般的に控えめな翻訳抑制15,31 &lt;をもたらすことが理解されています/ SUP&gt;、miRNA調節の主な役割は、タンパク質の出力を微調整し、遺伝子発現の異常なレベルから保護し、細胞の特定のプログラム32,33にロバスト性を提供することであることを示唆しています。 3&#39;LIFE画面における負のmiRNA / mRNAの相互作用の本質的に多数と組み合わせルシフェラーゼアッセイの感度は、多数の遺伝子に標的化miRNAの微妙な効果の検出を可能にし、その遺伝子ネットワークの複数のコンポーネントの識別与えられたmiRNA 24によって規制されています。 ここでは、3&#39;LIFEプロトコルを記述し、275人の3&#39;UTRを（ 図1）のパネルに対して2つの十分に特徴付けられたmiRNA、 のmiR-10Bおよびlet-7cとをスクリーニングすることによって、それが実現可能だ実証します。

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glycylglycine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">G1127-25G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kx PO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P2222</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E3889</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ATP</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">FLAAS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D0632</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C4282</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luciferin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">L9504</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S7653</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2EDTA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">E0399</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K H2 P O4</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">1551139</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A2153</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaN3</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">S2002</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coelenterazine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C3230</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS/HEPES</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21-040-CV</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D5546</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F2442</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pennicilin/Streptomicin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4333</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:71px;"></td>
			<td style="width:119px;">T2600000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Consumables</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MaxiPrep Kit</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">A2392</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well miniprep plate</td>
			<td style="width:71px;">Pall</td>
			<td>8032</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well shuttle plates</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">V4SP-2096</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5x Lysis Buffer</td>
			<td style="width:71px;">Promega</td>
			<td>E1941&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instruments</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well GloMax Plate Reader</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">E9032</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biomech FX Liquid Handler Robot</td>
			<td style="width:71px;">Beckmann</td>
			<td style="width:119px;">A31842</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4D-Nucleofector Core Unit</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">AAF-1001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well Shuttle System</td>
			<td style="width:71px;">Lonza</td>
			<td style="width:119px;">AAM-1001</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Counter Countess</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C10227</td>
		</tr>
	</tbody>
</table>

detection of mirna targets in high-throughput using the 3'life assay

の3&#39;UTR中の機能要素の発光識別（3&#39;LIFE）は、照会の3&#39;UTRの何百もの配列内の特定のmiRNAの標的の迅速な同定を可能にします。標的の同定は、miRNAが標的との機能的読み出しを与え、翻訳出力を測定することによって、mRNAレベルでの結合を検出するデュアルルシフェラーゼアッセイに基づいています。 3&#39;LIFEが可能でコスト効率のゲノムワイドな画面を作り、非独占バッファーと試薬、および公的に利用可能なレポーターライブラリを使用しています。 3&#39;LIFEは、標準的な実験室の設定のいずれかで行うか、液体処理ロボットや他の高スループットの計測器を使用してスケールアップすることができます。私たちは人間の96ウェルプレート中でクローニングの3&#39;UTR、および2つのテストのmiRNAのデータセットを使用してのアプローチを例示し、及びmiR-10B-7Cてみましょう 。我々は、96ウェルフォーマットにおいてDNAの調製、トランスフェクション、細胞培養およびルシフェラーゼアッセイを行い、データのためのツールを提供する方法を示し分析。結論3&#39;LIFEで体系的、迅速、高い再現性であり、かつ高い信頼性目標を識別します。

照度計の出力ファイルには、ホタルおよびウミシイタケルシフェラーゼタンパク質の両方の生の測定値が含まれています。と- MangoneラボのWebサイト（から入手可能な「3&#39;LIFE多分析」スプレッドシート-この生の形式は、「単板分析3&#39;LIFE」と互換性があり<a href="http://www.mangonelab.com">www.mangonelab.com</a> ）。単板解析スプレッドシートを自動的にホタル/ ウミシイタケ比を計算し、...

Watch this Scientific Journal Video about Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay at JoVE.com

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

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

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

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

detection-of-mirna-targets-in-high-throughput-using-the-3-life-assay

Research

JoVE Journal

Biology

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

10.0K ビュー.  Arizona State University. Luminescent identification of functional elements in 3’ untranslated regions (3’UTRs) (3’LIFE) is a technique to identify functional regulation in 3’UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

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

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 ...

生物学

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

ミッションターゲットIDのライブラリを使用してmiRNAのターゲットのゲノムワイドスクリーン

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

自動化を使用して染色体のハイスループット物理マッピング<em&gt;その場で</em&gt;ハイブリダイゼーション

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

ゲノムワイドな遺伝子欠失で<em&gt;ストレプトコッカス血統</em&gt;ハイスループットPCRによる

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

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 Functional Screening using a Homemade Dual-glow Luciferase Assay

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

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

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

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

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 ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

ハイスループットスクリーニングを使用したキナーゼ基質ペアの同定


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

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 ...