このプロジェクトは、サミュエル·ロバーツノーブル財団によって資金を供給された。著者はれたMSSを感謝します。アートワークのためのさんとケイティ·ブラウン;ジェイニーGallawayとコリーンElles優れた植物のケアのため。またれたMSSを感謝したいと思います。このプロトコルの確立時の技術的なサポートがトリーナコットレル、プージャUppalapati、Moumitaサハ、Swethaビヌコンダ氏アイザックGreenhut。

1。植物の成長とターゲット遺伝子サイレンシング<ol><li>植物の成長条件： <ol><li> Nをまくbenthamianaのは、土壌レスポッティング混合、メトロミックス350上の種と成長室に種を発芽。他の土壌または土壌レス媒体はまた、メトロミックスの代わりに使用することができる。 </li><li>移植3週齢の個々のポットに苗と21で維持温室でそれらを成長±2℃以前の文献12に記載されているような他の成長条件と一緒に。 2〜3日移植後に、植物をTRV接種に使用することができる。 </li></ol></li><li> TRV2クローンを育てる： TRVは二部ウイルスであり、そのゲノムはRNA1とRNA2から構成されています。 RNA1は、RNA依存性RNAポリメラーゼおよび移行タンパク質をコードする20,21。 RNA2は、コートタンパク質（CP）とサブゲノムRNAを21から2非構造タンパク質をコードしている。 RNA1とRNA2両方が成熟VIRの形成のために必要とされる私たちは、粒子とそのまん延20,21。 TRV2ベクター中のcDNAライブラリーの構築は、以前の文献22,23に記載されている。簡単に述べると、本研究で用いたVIGSライブラリは、様々な生物及び非生物的エリシターに露出葉組織から抽出したRNAから構築された。 <ol><li>冷凍庫からTRV2ベクトル（96ウェルプレート）でcDNAクローンを含むアグロバクテリウム （GV2260ひずみ）を取り出す。徐々にそれを解凍し、文化が室温に達した後、96ピンレプリケータを使用リファンピシン（10μgの/ ml）とカナマイシン（50μgの/ ml）でルリア-ベルターニ（LB）寒天プレート上に個々のアグロバクテ文化を接種する。 </li><li>二日間28℃でプレートをインキュベートする。十分なアグロバクテリウムの接種が2植物のプリック接種のために利用可能ですので、我々は通常、各クローンのための4つの複製コロニーを成長させる。無菌条件下ですべての手順を実行します。 </li></ol></li><li> VIGS： <ol><li>育つ28でTRV1を運んでアグロバクテリウム （GV2260）°上記の抗生物質を含むLB液体培地でC。一晩増殖させ培養液から遠心分離により細胞を回収し、接種緩衝液（10mM MES、pHが5.5; 200μMアセトシリンゴン）に再停止し、50 rpmでシェーカー上で、室温で3時間インキュベートする。 </li><li>遠心分離により細胞を回収し、5 mMのMES緩衝液（pH 5.5）及び接種（OD 600 = 0.3）に再懸濁する3-4 Nの背軸側へbenthamianaのは、無針注射器を使用して残します。詳細な手順は、接種前の文献24に示されている。その後、TRV1接種部位で、つまようじで葉を刺すことによって、それぞれのTRV2コロニーを接種する。 </li><li>活発な成長が効率的VIGS 12のために重要であるとして、十分な栄養と植物を維持する。約2週間、標的遺伝子の転写産物を減らすために開始され、植物が病原菌INOCの準備ができているポスト接種TRV接種後3週でulation。 </li></ol></li></ol> 2。非宿主病原体培養と植物接種の調製<ol><li>本研究で用いた病原体の詳細： シュードモナスシリン PV。 コナジラミ、P.シリン PV。 トマトT1、P. syringaeのPV。glycineaと白葉。vesicatoria、本研究で使用されています。 P.を育てるキングスB（KB）12時間28℃でリファンピシン（10μgの/ ml）を、カナマイシン（50μgの/ ml）で補足液体培地でシリン系統。 X.を育てる16時間LB液体培地で白葉。vesicatoria。すべての病原体は以前の文献19に記載されているようにGFPuvのを表現することができるプラスミドを抱く。 </li><li>植物の接種のために病原体培養液の調製： <ol><li>遠心分離により菌体を採取し、長いWAを使って緑色蛍光の存在を確認暗闇の中でvelength UVランプ。滅菌水で2回細胞を洗浄し、それらは、滅菌水を使用して所望の濃度に再懸濁する。 </li><li>スポット（約1.5cm直径）として標的遺伝子サイレンシングの葉（ 第 5〜8 番目 ）の背軸側にそれぞれの病原体（複数可）を接種する。いくつかの非宿主病原体が同時に標的遺伝子サイレンシング植物におけるそれらの成長のために試験することができる。これは植物体細菌の増殖を表示するための陽性コントロールとして用いることができると同時に、ホスト病原体を接種する。また、ベクトル制御（TRV :: 00）植物を接種。 </li></ol></li></ol> 3。細菌性病原体の植物体の成長での観察<ol><li>暗闇の中で長波長のUV光に接種葉を公開します。細菌のコロニーを葉の表面から放出される赤色蛍光のバックグラウンドでの葉の背軸側に緑色蛍光ドットとして表示することができます。 </li><li>病原体の成長を観察する2日後に接種（dpi）で5〜解像度。この時間間隔の間、毎日観察が必要である。 </li><li>後の最初の画面、短いリストサイレン一つ以上の非宿主病原体（複数可）の成長の結果クローン。 </li><li>選択されたクローンのためVIGSを繰り返し、再び非宿主病原体（S）への遺伝子沈黙植物の応答をテストします。スクリーニングのこの第二のレベルは、最初の画面の間に得られた偽陽性を取り除くために行われます。 </li></ol> 4。危殆非宿主抵抗のため選考植物の確認<ol><li>上記のような画面から選択されたcDNAクローンを沈黙。 5分 - 0.01％とそれぞれの病原体培養（v / v）のシルウェットL-77と植物を沈めると、3のために真空に沈水植物を施すことにより非宿主病原体（S）と植物全体に接種。後述するように、従来の増殖アッセイ6,18,19により細菌の増殖を定量化する。 </li><li> 0時間後に接種（HPI）で、3解像度と5円から、リーフパンチ（0.5 cm 2で ）を使って5生物学的には、各非宿主病原体（s）のために複製接種葉から2葉のサンプルを収集。葉のサンプルを、2日間28℃で適切な抗生物質やインキュベートを補っKB寒天培地上のシリアル希釈し、プレート樹液を挽く。コロニーカウンターを用いて細菌のコロニーをカウントし、前の文献6に記載されているように葉の中の細菌の成長を計算します。 </li><li>非宿主病原体（S）の影響を受けやすい植物は、ベクトル制御に比べて病原体の成長より高い数値を示すべきである。 </li></ol> 5。標的遺伝子の挿入と識別をシーケン<ol><li> attB1とattB2プライマーを用いたりTRV2ベクター内クローニング部位に隣接するプライマーを使用して、選択したクローンについてコロニーPCRを行います。ゲル上でPCR産物を実行し、単一バンドの増幅を確認してください。 </li><li> TRV2ベクター中の植物遺伝子インサートのattBを用いて配列決定することができるプライマーまたはクローニング部位に隣接するプライマー。 </li><li>シーケンスを使用してBLASTを実行し、遺伝子の詳細情報を識別します。 </li><li>非翻訳領域（UTRを）とともに完全長遺伝子配列を取得します。 </li><li>シーケンスの三つの異なる領域が300から400 bpの断片を選択し、TRV2ベクターにそれらのクローンを作成。独立して3つのすべてのVIGSコンストラクトを使用してVIGSを実行し、すべての3つの遺伝子の沈黙植物の非宿主抵抗性の妥協点を確認してください。 </li></ol>

<ol>
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我々は、開示することは何もありません。

植物免疫はほとんどまたは全く緑色蛍光がベクトル制御プラントから放出されていない、したがって、非宿主病原体の成長を制限し、長波長のUV光（ 図3D）の下で非宿主病原体を接種した葉。しかし、非宿主抵抗性に関与する遺伝子が沈黙したとき、遺伝子沈黙植物は非宿主病原体と緑色蛍光の成長（ 図3E）見られているを好む。これは、この原稿に記載された方法?...

非宿主抵抗は、特定の病原体1,2のレースに対するすべての植物種の抵抗である。これは、広いスペクトルおよび植物2,3における耐久性耐病性を付与する。しかし、特に細菌の病原体に対するそのメカニズムは、ウェル4を理解されていない。変異または沈黙植物その妥協非宿主抵抗性、および非宿主抵抗5-9間に差次的に発現する遺伝子を同定するためのハイスループット転写プロファイリングのためのスクリーニングは以前に細菌の非宿主抵抗性を解剖するために使用される2つの主要なアプローチである。非宿主抵抗が多くの遺伝子、遺伝子同定のためのハイスループット機能的ゲノムアプローチの関与を持つ複雑なメカニズム（S）4によって制御されているので、非宿主抵抗機構（s）を理解し、より良いために重要です。 ウイルス誘発性遺伝子サイレンシング（VIGS）が成功裏に内因性の植物を沈黙するのに使用されています多くの植物種の遺伝子10,11。 ベンサミアナタバコは VIGS 10,12に最適な植物の一つで、そのドラフトゲノム配列は現在13入手可能です。 たばこガラガラウイルス （TRV）ベースVIGSが広く逆遺伝学ツールとして使用されてきた非宿主抵抗2,4,14に関与する遺伝子を特徴づける。このVIGSベクトルと誘導体はシロイヌナズナの生 ​​物遺伝資源センター（ABRCを通じて利用可能になりました<a href="http://www.arabidopsis.org/abrc/catalog/individ_cloned_gene_1.html" target="_blank">http://www.arabidopsis.org/abrc/catalog/individ_cloned_gene_1.html</a> ）。 VIGSはまた、植物免疫15-17、特に非宿主抵抗6,18に関与する遺伝子を同定するための順方向遺伝学ツールとして使用されている。過敏感反応（HR）媒介性の非宿主特異的病原体に対する植物によって誘導される細胞死を評価し、疾患誘発される細胞死を評価することidentifyin主に使用される2つの主要なアッセイであるgの影響を受けやすい遺伝子は、植物を沈黙。しかし、HR細胞死は、タイプ-IIの非宿主病原体に対するとしないタイプ-I非宿主病原体2に対して誘導される。したがって、HRアッセイは普遍的に、特に、I型非宿主病原体の広い範囲に対して、プラントで使用される非宿主抵抗戦略を識別するために使用することができない。また、遺伝子の沈黙植物における非宿主抵抗性の部分的な損失は、常に病気の症状6ひいては病気のスコアが非宿主抵抗性を損なう植物を識別するために使用することはできないにつながるものではない。逆に、遺伝子沈黙植物の非宿主病原体の成長を評価する遺伝子沈黙植物の非宿主抵抗性の損失を研究するための良い方法です。 従来の増殖アッセイ6,19に比べて、遺伝子サイレンシング植物に非宿主細菌の増殖を評価するための高速な方法は、フォワード遺伝スクリーニングに要する時間を短縮することができる。我々は以前にバクテリアを観察するための方法を報告紫外線の下で肉眼での葉の上にL病原体の成長（UV）は、ライトグリーン蛍光タンパク質（GFP） を発現している19細菌を用いた。この原稿では、非宿主抵抗を侵害された遺伝子沈黙植物の識別を容易にするための非宿主細菌性病原体を表現GFPuvのの有用性を実証する。この方法は、感受性植物を同定するための正確かつハイスループットスクリーニングに適している。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>96-well U-bottom plates</td>
 <td> Becton Dickinson Labware (Franklin Lakes, NJ, USA)</td>
 <td> 35-3077</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>96-pin replicator stainless steel</td>
 <td> Nalge Nunc International (Naperville, IL, USA)</td>
 <td> 250520</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> High Intensity UV Inspection Lamps, Model B-100ap</td>
 <td> Thomas scientific (Swedesboro, NJ, USA)</td>
 <td> 6283K50</td>
 <td> Manufacturer ID 95-0127-01</td>
 </tr>
 <tr>
 <td> Stuart SC6 colony counter</td>
 <td> Bibby Scientific Limited, Staffordshire, UK</td>
 <td> SC6PLUS</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Soil-less potting mixture, Metro-Mix 350</td>
 <td> SUNGRO Horticulture Distribution, Inc., (Bellevue, WA, USA)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Primers: 
 attB1 (GGGGACAAGTTTGTACAAAAAAGCAGGCT) 
 attB2 (GGGGACCACTTTGTACAAGAAAGCTGGGT)</td>
 <td> Integrated DNA Technologies, Inc (Coralville, IA, USA)</td>
 <td> Custom synthesized</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> MES, Monohydrate</td>
 <td> VWR international (Radnor, PA, USA)</td>
 <td> CAS No. 145224-94-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Acetosyringone (Dimethoxy-4'-hydroxyacetophenone)</td>
 <td> Sigma Aldrich (St. Louis, MO, USA)</td>
 <td> D134406</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> Vac-In-Stuff (Silwet L-77)</td>
 <td> Lehle Seeds (Round Rock, TX, USA)</td>
 <td> VIS-30</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

vigs-mediated forward genetics screening for identification of genes involved in nonhost resistance

細菌性病原体に対する植物の非宿主病抵抗性は、複雑な防御経路によって制御される。このメカニズムを理解することは、病原体の広い範囲に対して耐久性のある病害抵抗性植物を開発するために重要である。ウイルス誘発性遺伝子サイレンシング（VIGS）ベースのフォワード遺伝スクリーニングは、非宿主抵抗性を付与する植物防御遺伝子の同定に有用なアプローチである。 タバコガラガラウイルス （TRV）ベースVIGSベクトルは、現在までに、最も効率的なVIGSベクトルでかつ効率的に使用されていベンサミアナタバコにおける内因性標的遺伝子をサイレンシングする。 本稿では、我々はNのcDNAライブラリーからの個々のクローンのサイレンシングのために前方の遺伝スクリーニングアプローチを実証ベンサミと非宿主病原体に対する妥協非宿主抵抗性の遺伝子沈黙植物の応答を評価し、 シュードモナスシリン PV。 トマトT1、P. syringaeのPV。glycinea、およびX.白葉。vesicatoria。これらの病原菌は、GFPuvのタンパク質を発現するように設計されており、標的遺伝子サイレンシングが非宿主性を付与に関与している場合、それらの緑色蛍光コロニーを接種した植物の非宿主病原体にUV光の下で肉眼で見ることができる。これは、非宿主病原体に感受性遺伝子沈黙植物の安定した高速の識別を容易にします。さらに、有望な候補遺伝子情報はTRVベクター中の植物遺伝子挿入物を配列決定することによって知ることができる。ここでは、非宿主抵抗性に関与する遺伝子を同定するためVIGS媒介フォワード遺伝学の高スループット能力を示しています。およそ、100 cDNAを個別に約2〜3週間で沈黙することができ、いくつかの非宿主細菌の病原体に対する非宿主抵抗性との関連性は、その後、週に研究することができる。本稿では、このスクリーニングに関わる詳細な手順を列挙。 VIGS媒介フォワード遺伝screeningのアプローチは、非宿主抵抗性に関与する遺伝子を特定するだけでなく、様々な植物種のいくつかの生物と非生物的ストレスの許容範囲を付与する遺伝子を研究するだけでなく、拡張することができます。

本研究の主要な目的は、Nを沈黙遺伝子を容易かつ正確に同定するための方法を実証することである非宿主抵抗性のために妥協しているベンサミ工場 。この方法論の4つの主要なステップがあります。最初のステップは、個別にTRV-VIGSを使用して多数の遺伝子を沈黙させることである。我々は、 図1に示されているプロトコルを使用して、約1.5年間にわたり約5,000遺伝?...

Watch this Scientific Journal Video about VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance at JoVE.com

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

ウイルス誘発性遺伝子サイレンシングは、植物の非宿主抵抗性に関与する遺伝子を同定するための便利なツールです。私たちは、非宿主病原体に感受性遺伝子沈黙植物を識別するのにGFPuvのを表現する細菌の病原体の使用方法を示しています。このアプローチは速く、容易であり、大規模なスクリーニングと同様のプロトコルは、様々な他の植物、微生物の相互作用を研究に適用することができる容易にする。

非宿主抵抗性に関与する遺伝子の同定のためのVIGS媒介フォワード遺伝スクリーニング


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

vigs-mediated-forward-genetics-screening-for-identification-genes

Research

JoVE Journal

Immunology and Infection

16.9K ビュー.  The Samuel Roberts Noble Foundation. ウイルス誘発性遺伝子サイレンシングは、植物の非宿主抵抗性に関与する遺伝子を同定するための便利なツールです。私たちは、非宿主病原体に感受性遺伝子沈黙植物を識別するのにGFPuvのを表現する細菌の病原体の使用方法を示しています。このアプローチは速く、容易であり、大規模なスクリーニングと同様のプロトコルは、様々な他の植物、微生物...

ビデオ: VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in bioterrorism is also a concern. A better understanding of the cellular factors that impact infection would facilitate the development of much-needed therapeutics. Recent advances in RNA interference (RNAi) technology coupled with complete genome sequencing of several organisms has led to the optimization of genome-wide, cell-based loss-of-function screens. Drosophila cells are particularly amenable to genome-scale screens because of the ease and efficiency of RNAi in this system 1. Importantly, a wide variety of viruses can infect Drosophila cells, including a number of mammalian viruses of medical and agricultural importance 2,3,4. Previous RNAi screens in Drosophila have identified host factors that are required for various steps in virus infection including entry, translation and RNA replication 5. Moreover, many of the cellular factors required for viral replication in Drosophila cell culture are also limiting in human cells infected with these viruses 4,6,7,8, 9. Therefore, the identification of host factors co-opted during viral infection presents novel targets for antiviral therapeutics. Here we present a generalized protocol for a high-throughput RNAi screen to identify cellular factors involved in viral infection, using vaccinia virus as an example.

RNAi Screening for Host Factors Involved in Vaccinia Virus Infection using Drosophila Cells

Novel host factors involved in viral infection can be identified through cell-based genome-wide loss of function RNAi screening. A Drosophila cell culture model is particularly amenable to this approach due to the ease and efficiency of RNAi. Here we demonstrate this technique using vaccinia virus as an example.

に関与する宿主因子のためのRNAiスクリーニングワク</em使用して>ウイルス感染ショウジョウバエセル

Viral pathogens represent a significant public health threat; not only can viruses cause natural epidemics of human disease, but their potential use in ...

免疫・感染学

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

ヒトのインビトロ</em免疫バランスの初期状態の認識のためのスクリーニングツールとして>抑制

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

感染マウスノロウイルスのリバースジェネティクスを介したリカバリ

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

フォワード遺伝的アプローチで<em&gt;クラミジアトラコマチス</em

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

アルギン酸塩産生に関連する新規遺伝子の同定<em&gt;緑膿菌</em使用&gt;ミニ<em&gt; himar1</em&gt;マリナートランスポゾンによる突然変異誘発

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆転写酵素及びインテグラーゼ阻害剤の迅速なスクリーニング

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&amp;#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

フォワード遺伝学は、識別するためのマクロファージを使用した画面<em&gt;トキソプラズマ</emIFN-γ依存性細胞自律イミュニティへの抵抗のための重要な&gt;遺伝子

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections.
Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring.
In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells.
In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular bacteria based on the GFP signal, with only intracellular bacteria being able to express GFP. This allows the robust detection of single intracellular bacteria before intracellular proliferation is initiated.

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells

Two assays for microscopy-based high-throughput screening of host factors involved in Brucella infection are described. The entry assay detects host factors required for Brucella entry and the endpoint assay those required for intracellular replication. While applicable for alternative approaches, siRNA screening in HeLa cells is used to illustrate the protocols.

に関与する宿主因子のハイスループットスクリーニングのための顕微鏡ベースのアッセイ<em&gt;ブルセラ</em&gt; HeLa細胞の感染

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.