ホワイトヘッド研究所フェローファンド

パート1：感染症のセットアップ<ol><li>フラスコにHeLa細胞を成長し、細胞が約80％コンフルエントになるまで待ちます。 </li><li> 2％FBSとの定期的なDMEMと添加しない抗生物質：実験のために十分なウイルスの増殖培地を準備します。 </li><li>感染症は、どちら原油既知の力価を持つワクシニアウイルスの株式、またはあなたがホストの遺伝子の発現が心配な場合は、既知の力価でウイルスを精製白糖を行うことができます。 </li><li>あなたがウイルスを精製白糖を使用している場合は、パート2に直接進みます。 </li><li>あなただけの感染前に、原油ワクシニアウイルス株を使用している場合は、主に氷のお風呂と少量の水でカップの超音波処理でウイルスのアリコートを超音波で分解する。 <ul style="list-style-type:lower-alpha;"><li> 20秒のために30W前後で超音波処理。 </li><li>ボルテックスチューブと氷とチルドを充填したカップを保つために、必要に応じてより多くの氷を追加し、超音波処理を3回繰り返す。 </li><li>各超音波処理のステップの間に渦。 </li><li>パート2に進んでください。 </li></ul></li><li>また、あなたがカップの超音波処理を有していない場合、原油ウイルスストックと0.25mg/mLのトリプシンの等量を混合する。 <ul style="list-style-type:lower-alpha;"><li>激しくボルテックスする。 </li><li> 30分間37℃ウォーターバスでのウイルスストック/トリプシンミックスをインキュベートする。 </li><li> 5〜10分間隔で渦。 </li><li>パート2に進んでください。 </li></ul></li></ol>パート2：細胞に感染<ol><li>感染の目的の多重度（MOI）で単層に感染するために必要なウイルス粒子の量を計算します。一般的に、MOI（5〜10）高は、感染のtimecoursesに使用されます。 </li><li> 37ウイルスまたはトリプシン処理原油のウイルスを精製白糖、所望量の° Cのウイルス増殖培地を加え、よく混ぜる。あなただけのフラスコの底部をカバーするのに十分なメディアを使用する必要があります。たとえば、T - 175フラスコのためのメディアを10mL。 </li><li>細胞から培地を取り除き、室温のPBSですすいでください。 </li><li>各フラスコにウイルス/ウイルス増殖のメディアを追加します。 <ul style="list-style-type:lower-alpha;"><li>優しくスワール、37℃傾斜プレートをインキュベート℃で1時間5％CO2インキュベーターでC。 </li><li>チルトと渦のプレートは15​​分ごとに一様にウイルスを拡散し、細胞の潤いを保つために。 </li><li> 0hr/Mockの時点では、唯一のウイルスの増殖培地を追加し、ないウイルスには、追加された。 </li></ul></li><li> 1時間のインキュベーションの後、ウイル​​スを含むメディアを削除し、そして室温PBSで3回リンス。 </li><li>各フラスコにウイルスの増殖培地の最大量を追加。たとえば、T - 175フラスコのためのメディアの30 mLの。あなたの0時間の時点としてこれをカウントを開始。 </li></ol>パート3：収穫の細胞<ol><li>顕微鏡下で細胞を確認し、すべての細胞変性効果（CPE）を注意してください。 </li><li>メディアを取り出し、室温PBS 30 mLで細胞をリンス。 </li><li>細胞にトリプシンの15 mLを加え、37℃で2 - 5分間インキュベート℃に</li><li>細胞の剥離を顕微鏡で確認し、細胞を取り除くために、静かにフラスコをタップします。 </li><li> 50 mLコニカルチューブに滅菌血清ピペットで細胞を移す。 </li><li>細胞増殖培地の等量でフラスコを洗い、円錐管にトリプシン処理細胞にメディアを追加。 </li><li>室温で5分間、300 × gで細胞を遠心分離します。 </li><li>細胞ペレットからトリプシン/メディアを取り出します。 </li><li>ご希望のRNA単離キットからTRIzol試薬または溶解バッファーのいずれかで細胞ペレットを再懸濁します。 </li><li>あなたがTRIZOLを使用している場合は、-80℃で1.5mlのラベルが付いたエッペンドルフチュー​​ブや凍結の1mLのアリコートにサンプルを分割℃に</li><li>タイムポイントごとに1つのフラスコを収穫する、すべての時間ポイントについて繰り返します。 </li></ol>パート4：TRIZOLのサンプルのRNA抽出<ol><li>この時点で、あなたのサンプルを処理できる状態にTRIzol試薬中に再懸濁させ、されるべきである。 </li><li>各チューブのTRIZOLのすべての1 mLのためのBCPの相分離試薬の200μLを追加。あなたがTRIZOL大量の話から始めている場合、より大きなチューブにサンプルを転送する必要がある場合があります。 </li><li>ボルテックスや激しい振とうし、2〜3分間室温でインキュベートする。 </li><li> 15分間12,000 xgでサンプルを遠心分離します。 </li><li>新しいチューブに水相を移す。水相は、上に透明層である。あなたがから始めたTRIZOLのすべての1mLのための約600μL回復されるべきである。 </li><li>代わりに、BCPの第二のフェノール/クロロホルム抽出、1mlのトリゾールあたりのクロロホルム500μLを使用して、この時間を、ください。 </li><li>ボルテックスや激しい振とうし、2〜3分間室温でインキュベートする。 </li><li> 15分間12,000 xgでサンプルを遠心分離します。 </li><li>新しいチューブに水相を移す。水相は、上に透明層である。 </li><li>各サンプルに線形アクリルアミドの20μgを追加。リニアアクリルアミドは、キャリアとして機能し、RNAを沈殿させるために役立ちます。 </li><li> 500＆マイルを追加CRO、TRIZOL 1mlあたりイソプロパノールのLは、それぞれのサンプルにしてスタートし、よく混ぜる。 </li><li>室温で10分間サンプルをインキュベートする。 </li><li> 10〜15分、最高速度で遠心機で遠心する。 </li><li> RNAペレットは、チューブの下部に表示されます。非常に慎重に、どちらかの真空吸引器で、または手動ピペッターで、チューブから上清を取り除く。ペレットを乱さないでください。 </li><li> 70％エタノール1mLでペレットを洗浄します。 <ul style="list-style-type:lower-alpha;"><li>ペレットに70％エタノール1mLを追加。 </li><li>トップスピードで5-7分、14,000 gで遠心します。 </li><li>非常に慎重に、真空吸引器のいずれかで、チューブからエタノールを削除する、または手動ピペッターで。その際、ペレットがチューブの下部に表示されていることを確認してください。 </li><li>上清を捨てる。 </li></ul></li><li>洗浄ステップを繰り返します。上清を廃棄した後に、ペレットがチューブ上のどこにマークします。 </li><li> 5分よりもはや自然乾燥ペレット。乾燥し過ぎたしない、またはRNAを再構成することが難しくなるためです！ </li><li>あなたがサンプルから、残りのDNAを除去するために、オプションのDNase処理をフォローしたい場合は、ヌクレアーゼフリー水17μLでペレットを再懸濁します。そうでない場合は、ヌクレアーゼフリー水20μLでペレットを再懸濁し、22に進みます。 </li><li> （オプションのDNase処理）キアゲンのRNaseフリーのDNaseセットを使用して、2μlのバッファRDDと1μLで再懸濁したRNAにRNaseフリーのDNase Iを再構成に追加します。穏やかに混合する。 </li><li> （オプションのDNase処理）37℃で30分間。 </li><li> （オプションのDNase処理）65で2.5 mMのEDTAとインキュベートの2μlの追加℃で5分間は、DNaseを失活させる。長い時間/より高い温度ではRNAの分解を引き起こすことができるように不活性化の時間や温度を超えないようにしてください。 </li><li>分光光度計によるRNA濃度を確認してください。 </li><li> -80℃でのRNAサンプルを保存する</li><li> （オプションQC）アジレントバイオアナライザを用いてRNAの品質をチェック、または変性ゲルでサンプルを実行し、OD測定値をチェックして。 （260nmのと280分の260比） </li></ol>

<ol>
	<li>KH, R. u. b. i. n. s., LE, H. e. n. s. l. e. y., GW, B. e. l. l., Wang, C., EJ, L. e. f. k. o. w. i. t. z., PO, B. r. o. w. n., DA, R. e. l. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+viral+gene+expression+programs+during+poxvirus+infection:+a+transcriptional+map+of+the+vaccinia+and+monkeypox+genomes.">Comparative analysis of viral gene expression programs during poxvirus infection: a transcriptional map of the vaccinia and monkeypox genomes.</a> PLoS ONE. 3 (7), e2628-e2628 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+analysis+of+a+complete+poxvirus+transcriptome+reveals+an+immediate-early+class+of+genes.">Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes.</a> Proc. Natl. Acad. Sci. 105 (6), 2140-2145 (2008).</li><li>Satheshkumar, P. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poxvirus+transcriptome+analysis.">Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E62-E62 (2008).</li><li>Assarsson, E., Greenbaum, J. A., Sundstr&#246;m, M., Schaffer, L., Hammond, J. A., Pasquetto, V., Oseroff, C., Hendrickson, R. C., Lefkowitz, E. J., Tscharke, D. C., Sidney, J., Grey, H. M., Head, S. R., Peters, B., Sette, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.+Reply+to+Satheshkumar+and+Moss:+Poxvirus+transcriptome+analysis.">A. Reply to Satheshkumar and Moss: Poxvirus transcriptome analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 105, E63-E64 (2008).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., Mu&#241;oz, M., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+gene+expression+survey+of+vaccinia+virus+infection+of+human+HeLa+cells.">Cellular gene expression survey of vaccinia virus infection of human HeLa cells.</a> J Virol. 77 (11), 6493-6506 (2003).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Conde, R., Pascual-Montano, A., Harshman, K., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+reveals+characteristic+changes+of+host+cell+gene+expression+in+response+to+attenuated+modified+vaccinia+virus+Ankara+infection+of+human+HeLa+cells.">Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.</a> J Virol. 78 (11), 5820-5824 (2004).</li><li>Guerra, S., L&#243;pez-Fern&#225;ndez, L. A., Pascual-Montano, A., N&#225;jera, J. L., Zaballos, A., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+response+to+the+attenuated+poxvirus+vector+NYVAC:+upregulation+of+apoptotic+genes+and+NF-kappaB-responsive+genes+in+infected+HeLa+cells.">Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.</a> J Virol. 80 (2), 985-998 (2006).</li><li>Guerra, S., N&#225;jera, J. L., Gonz&#225;lez, J. M., L&#243;pez-Fern&#225;ndez, L. A., Climent, N., JM, G. a. t. e. l. l., Gallart, T., Esteban, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+gene+expression+profiling+after+infection+of+immature+human+monocyte-derived+dendritic+cells+by+the+attenuated+poxvirus+vectors+MVA+and+NYVAC.">Distinct gene expression profiling after infection of immature human monocyte-derived dendritic cells by the attenuated poxvirus vectors MVA and NYVAC.</a> 81 (16), 8707-8721 (2007).</li></ol>

重要なステップパート1＆2 ウイルス粒子を分解するために、同期ワクシニア感染症、ウイルスの最初のもの慎重に超音波処理を（またはtrypzinizing）、セットアップするには、いくつかの重要なステップがあります。ワクシニアウイルスは、集約の影響を受けやすい国である、とウイルス粒子の破壊は細胞のさらに感染を確保するために重要です。同期感?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>TRIzol Reagent</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>Similar reagents, such as TriPure from Roche, will also work.</td>
</tr>
<tr>
<td>BCP Phase Separation Reagent</td>
<td>Reagent</td>
<td>Molecular Research Center</td>
<td>BP151</td>
<td>&nbsp;</td>
<tr>
<td>RNase-Free DNase Set</td>
<td>Reagent</td>
<td>Qiagen</td>
<td>79254</td>
<td>DNase treatment is an optional step.</td>
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vaccinia virus infection &amp; temporal analysis of virus gene expression: part 1

家族の<em&gt;ポックスウイルス科</em&gt;感染細胞の細胞質中に独占的に複製するウイルスを含む大規模な二重鎖DNAで構成されています。のメンバー<em&gt;オルトポックス</em&gt;属インクルード天然痘、人間の天然痘、サル痘、およびワクシニア（VAC）の原因物質、ウイルスの家族の原型の一員。比較的大きな（〜200 KB）ワクシニアゲノムの中で、遺伝子の3つのクラスは、エンコードされています：初期、中間、および遅い。すべての3つのクラスがウイルスによりコードされたRNAポリメラーゼによって転写されていますが、各クラスには、ウイルスのライフサイクルの異なる機能を果たします。ポックスウイルスは感染時に宿主細胞環境の変調のための複数の戦略を利用する。両方のホストとウイルスの遺伝子発現の調節を理解するために、我々は、ウイルスと宿主細胞の両方から転写産物量を分析するためにゲノムワイドなアプローチを利用してきた。ここで、我々は、いくつかの時点感染後におけるワクシニアウイルスとサンプリングのRNAとHeLa細胞の経時的感染を示す。両方のホストとウイルスの全RNAを単離し、遺伝子発現の解析用マイクロアレイへのハイブリダイゼーションのために増幅されます。

Watch this Scientific Journal Video about Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1 at JoVE.com

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 1

HeLa細胞と宿主とウイルスの遺伝子発現の解析のワクシニア感染のためのプロトコル。 3のパート1。

ワクシニアウイルス感染とウイルスの遺伝子発現の時間的解析：その1

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells.  Members of ...

vaccinia-virus-infection-temporal-analysis-virus-gene-expression-part

Research

JoVE Journal

Biology

10.2K ビュー.  MIT - Massachusetts Institute of Technology. HeLa細胞と宿主とウイルスの遺伝子発現の解析のワクシニア感染のためのプロトコル。 3のパート1。

ビデオ: Vaccinia Virus Infection & Temporal Analysis of Virus Gene Expression: Part 1

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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 2

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 2 of 3.

ワクシニアウイルス感染とウイルスの遺伝子発現の時間的解析：その2

Vaccinia Virus Infection &amp; Temporal Analysis of Virus Gene Expression: Part 3

The family Poxviridae consists of large double-stranded DNA containing viruses that replicate exclusively in the cytoplasm of infected cells. Members of the orthopox genus include variola, the causative agent of human small pox, monkeypox, and vaccinia (VAC), the prototypic member of the virus family. Within the relatively large (~ 200 kb) vaccinia genome, three classes of genes are encoded: early, intermediate, and late. While all three classes are transcribed by virally-encoded RNA polymerases, each class serves a different function in the life cycle of the virus. Poxviruses utilize multiple strategies for modulation of the host cellular environment during infection. In order to understand regulation of both host and virus gene expression, we have utilized genome-wide approaches to analyze transcript abundance from both virus and host cells. Here, we demonstrate time course infections of HeLa cells with Vaccinia virus and sampling RNA at several time points post-infection. Both host and viral total RNA is isolated and amplified for hybridization to microarrays for analysis of gene expression.

Protocol for Vaccinia infection of HeLa cells and analysis of host and viral gene expression. Part 3 describes the process of fluorescently labeling the amplified RNA from both host and viral samples by amino allyl coupling of dyes. Part 3 of 3.

ワクシニアウイルス感染とウイルスの遺伝子発現の時間的解析：その3

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

ウイルス誘導ジーンサイレンシング（VIGS）でタバコbenthamianaとトマト

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

コットンでアグロバクテリウムを介したウイルス誘発遺伝子サイレンシングアッセイ

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBLでMultiBacタンパク質複合体生産プラットフォーム

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...