私たちは、原稿上で彼女の貴重な洞察力のために博士パトリシアManosalvaに感謝。資金は、全米科学財団の植物ゲノムのプログラム、受賞番号DBI - 0605059で提供されていました。

パート1：植物材料サイレンシングのために使用されるN. benthamiana植物は2前後となるはず½子葉や最初の2時間である週は古い- 4本葉が浮上している。 8日間の後出現、真の葉はまだ登場していない-トマト（ トマト ）の植物は、7を使用されています。 パート2：VIGS 1日目<ol><li>各実験、pTRV1をかくまってアグロバクテリウムツメファシエンス 、pTRV2の場合は、pTRV2 - PDSとpTRV2 -ホストの標的遺伝子は、カナマイシン50μg/ mLのとリファンピシンの100μg/ mlのを補充したLB寒天プレート上で増殖されています。リファンピシンは、 アグロバクテリウムのためにそうする間、カナマイシンは、プラスミドpTRVのために選択されます。 2日間、30℃でインキュベートする。 PDSのサイレンシングは退色するために植物が発生し、サイレンシング効率制御として使用されます。また、ダウンレギュレートする遺伝子はpTRV2ベクターにクローン化されている必要があります。クローニングを容易にすることができるとLiuらによって記述されているゲートウェイの互換性のあるpTRV2ベクトルがあります。 （2002）。 </li></ol> 3日目<ol><li>菌株のそれぞれについて、上記の抗生物質とLBの3 mLの液体培養 - 2を接種する。 18時間、200 rpmの - 16、30℃で振とうしてインキュベートします。 </li></ol> 4日目<ol><li>カナマイシン、リファンピシンおよび200μMのアセトシリンゴン（表1、2、3）と二次液体誘導メディア（IM）、IMの文化に初代培養の25倍希釈：1接種する。アセトシリンゴンは、IMは、環境のホストのアポプラストにおけるこの病原体の出会いを模倣しながら工場9にT - DNAの伝達のために必要とされているアグロバクテリウムのvir遺伝子の誘導物質として使用されます。 20分間30℃で振とうしてインキュベートする - 24時間、200 rpmの</li></ol> 5日目<ol><li> 3000 × gで10分間centrifugatingして細胞を収集するオリジナルの文化が10mMのMgCl 2、10mMのMES pHを5.5に持っていたのと同じボリュームに再懸濁します。細胞を穏やかにそれらを再懸濁するためにボルテックスすることができます。 </li><li> 3000 xgで10分間、再び細胞をCentrifugue 10mMのMgCl2を、10mMのMES液（pH5.5）で元の培養液の半分量に再懸濁します。 </li><li>各細菌培養は、0.3のOD 600との細菌懸濁液を準備します。 pTRV1文化〜400μMの最終濃度にアセトシリンゴン追加。 </li><li> pTRV1と1対1の割合でpTRV2を（または目的の遺伝子を含むpTRV2）を含む培養液を混ぜる。またpTRV2 - PDSのコントロールが含まれています。最後のアセトシリンゴン濃度が現在は200μMであり、それぞれの文化が0.15のOD 600に位置していることに注意してください。 </li><li>沈黙する遺伝子と実験の日付を浸透する苗木にラベルを付けます。 </li><li>針を浸透するために、各葉に穴を開けない。苗に細菌懸濁液を浸透させるために1 mLの不必要な注射器を使用してください。トマトの場合は、Nの間に、両方の子葉に潜入benthamianaは 、最大のtwo真の葉に浸潤する。各細菌の混合物の5mlのは、15 N を浸透させるために十分なはずですbenthamianaと25トマトの苗。浸潤との間および接種後次の日までの植物の水遣りしないことにより、手袋を変更することにより、クロスコンタミネーションを避ける。 </li><li>植物は20℃に保たれている - 22 ° C 16時間の日長とそれらがアッセイに使用される前に、少なくとも3 ½の週間50％RHと成長チャンバーインチ</li></ol>パート3：代表的な結果図1は、Nと代表的な実験を示しています。 PDSのために沈黙benthamianaとトマト。植物は、カロテノイドの減少量と植物で観察される特徴的な退色表現型を示す。 PDS -サイレンシング制御の植物のため、浸潤後1 ½の週とすぐに見られるように開始を光退色。 <img src="/files/ftp_upload/1292/Figure 1A 1292.jpg" alt="図1a" /><img src="/files/ftp_upload/1292/Figure 1B 1292.jpg" alt="図1b" /> 図1。PDS制御遺伝子のサイレンシングは、N. benthamiana（A）とトマト（B）植物の光退色が発生します。写真は、3 ½の週間サイレンの後に撮影された。 表1。誘導培地（IM）の調製。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 400mLの</td><td>蒸留H 2 O </td></tr><tr><td> 4.88グラム</td><td> MES（2 - （4モルホリノ）エタンスルホン酸） </td></tr><tr><td> 2.5グラム</td><td>グルコース</td></tr><tr><td> 0.12グラム</td><td>のNaH 2 PO 4 </td></tr></tbody></table>のdH 2 Oで475 mlの最終体積に持参し、5.6にpHを調整する。オートクレーブ。培地冷却された後、20X AB塩の25 mLを加え。 表2。AB塩の調製。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 20グラム</td><td> NH 4 Clを </td></tr><tr><td> 6グラ​​ム</td><td>し、MgSO 4 · 7H 2 O </td></tr><tr><td> 3グラム</td><td>塩化カリウム</td></tr><tr><td> 0.2グラム</td><td> CaCl 2の </td></tr><tr><td> 0.05グラム</td><td>のFeSO 4 · 7H 2 O </td></tr></tbody></table>蒸留水で1リットルの最終容量にもたらす。オートクレーブ。 AB塩は橙色の粉末として沈殿させることに注意してください。ちょうどその使用前に渦巻くことでよく混ぜる。 表3。200mMのアセトシリンゴンの準備。アセトシリンゴンは、それが使用される日を準備する必要があることに注意してください。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 19.6ミリグラム</td><td>アセトシリンゴン（3&#39;、5&#39; -ジメトキシ-4&#39; - ヒドロキシアセトフェノン） </td></tr><tr><td> 500μL </td><td> DMSO（ジメチルスルホキシド） </td></tr></tbody></table>

<ol>
	<li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+as+a+plant+immune+system+against+viruses.">RNA silencing as a plant immune system against viruses.</a> Trends Genet. 17, 449-459 (2001).</li><li>Carrington, J. C., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microRNAs+in+plant+and+animal+development.">Role of microRNAs in plant and animal development.</a> Science. 301, 336-338 (2003).</li><li>Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microRNA+as+a+translational+repressor+of+APETALA2+in+Arabidopsis+flower+development.">A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development.</a> Science. 303, 2022-2025 (2004).</li><li>Palatnik, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+the+tobraviruses.">Molecular biology of the tobraviruses.</a> J. Gen. Virol. 80, 2799-2807 (1999).</li><li>Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+study+to+determine+the+extent+of+gene+silencing+in+Nicotiana+benthamiana+and+other+Solanaceae+species+when+heterologous+gene+sequences+are+used+for+virus-induced+gene+silencing.">A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing.</a> New Phytol. 176, 782-791 (2007).</li><li>McCullen, C. A., Binns, A. 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ウイルス誘発性遺伝子サイレンシングは、急速な逆遺伝学的スクリーンを可能にする方法です。それは、T - DNAまたはシロイヌナズナとトウモロコシのような特定の植物にのみ利用可能なトランスポゾン媒介遺伝子ノックアウトアウト、の生成が回避される。また、植物形質転換の時間のかかるプロセスを回避し、彼らが10またはその別のホストの標的配列を十分な相同性を持?...

virus-induced gene silencing (vigs) in nicotiana benthamiana and tomato

RNA干渉（RNAi）は、dsRNAの1によって引き起こさ非常に特異的な遺伝子サイレンシングの現象です。マイクロRNA、非蛋白質をコードする遺伝子と短い干渉RNA（siRNA）から生成されています：このサイレンシング機構は、RNAのレギュレータの2つの主要なクラスを使用しています。植物は、トランスポゾンを制御するため、および花の器官形成と葉の開発2,3,4などの発達過程を厳密にコントロールを発揮するために、RNAiを使用してください。植物はまた、ウイルスによる感染から身を守るために、RNAiを使用してください。その結果、多くのウイルスはその宿主5の彼らの成功した植民地化を可能にするためにサイレンシング遺伝子の抑制を進化させてきた。 ウイルス誘発性遺伝子サイレンシング（VIGS）は、植物RNAiによる抗ウイルス防御機構を利用する方法です。変更されていないウイルスに感染した植物では機構は、特にウイルスのゲノム比較対象としています。しかし、ホストの遺伝子由来の配列を有するウイルスベクターを用いて、プロセスはさらに、対応するホストのmRNAを対象とすることのできる。 VIGSは、プラスミド、そのチタン、全体またはサイレンシングの対象となる遺伝子配列の一部を担持する組換えウイルスを介して、配信するために植物病原体アグロバクテリウムツメファシエンスを使用して、植物におけるハイスループットゲノム機能解析に適応されています。全身性ウイルスの拡散と内生植物のRNAi機構は、残りの世話をする。標的遺伝子に対応するdsRNAは長さ21〜24ヌクレオチドのsiRNAへのリボヌクレアーゼのダイサーによって生成され、その後切断されています。これらのsiRNAは最終的にターゲット転写物2を分解するRNA誘導サイレンシング複合体（RISC）を導く。 異なるベクターはVIGSで採用されていると、最も頻繁に使用されるのいずれかがタバコ茎えそウイルス（TRV）に基づいています。 TRVは、二分ウイルスであり、そのような、二つの異なるAとしてツメファシエンス株は 、VIGSに使用されます。一つは、pTRV2、他のコートタンパク質とVIGS 6,7のために使用されるシーケンスを庇護しながら複製し、動きのウイルスの機能をコードpTRV1を、運びます。遺伝子サイレンシングの菌株の結果、両方の混合物とタバコbenthamianaとトマトの苗を接種。退色の原因内因性フィトエンデサチュラーゼ（PDS）遺伝子のサイレンシングは、VIGSの効率のためのコントロールとして使用されます。それはトマトのサイレンシングは通常Nに比べて非効率であること、しかし、注意すべきであるbenthamiana。目的の遺伝子のRNA転写物の豊富さは、常に、ターゲット遺伝子を効率的にダウンレギュレートされていることを保証するために測定する必要があります。 N.からそれにもかかわらず、異種遺伝子配列benthamiana、トマト、その逆も8で、それぞれのオルソログを黙らせるために使用することができます。

Watch this Scientific Journal Video about Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato at JoVE.com

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

ノックダウン遺伝子発現の内のウイルス誘発性遺伝子サイレンシング（VIGS）メソッドの説明<em&gt;タバコbenthamiana</em&gt;とトマト。

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 ...

virus-induced-gene-silencing-vigs-in-nicotiana-benthamiana-and-tomato

Research

JoVE Journal

Biology

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

52.4K ビュー.  Cornell University. ノックダウン遺伝子発現の内のウイルス誘発性遺伝子サイレンシング（VIGS）メソッドの説明タバコbenthamianaとトマト。

ビデオ: Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

siRNAを介した遺伝子サイレンシングと組み合わせることで生細胞のカルシウムイメージングでは、CAを識別 2 + ER膜のリークチャネルとその調節機序

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

生物学

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

細胞培養で誘導し、低酸素のテスト

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

ジャガイモとニコティアナベンタミアナにおけるアグロインフィルトレーションとPVXアグロパネクチ

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

最適化と利用の<em&gt;アグロバクテリウム</em中&gt;媒介一過性タンパク質産生<em&gt;タバコ</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

病気のベクトル蚊幼虫におけるキトサン/干渉RNAナノ粒子媒介遺伝子サイレンシング

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

siRNA媒介サイレンシングによるマウス卵母細胞における減数分裂紡錘体の評価

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

ベンサミアーナ タバコ葉蛋白質蛋白質の相互作用ダイナミクスの一過性の決定のための試金補完ルシフェラーゼ イメージング

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...