Nous remercions le Dr Patricia Manosalva pour ses précieuses sur le manuscrit. Le financement a été fourni par le Programme National Science Foundation génome des plantes, le nombre récompense DBI-0605059.

Partie 1: Les matières végétales N. benthamiana utilisées pour faire taire devrait être d&#39;environ 2 ½ semaines qui est le moment où les cotylédons et les 2 premiers - 4 vraies feuilles ont émergé. Tomate (Solanum lycopersicum) les plantes sont utilisées de 7 à 8 jours après l&#39;émergence, quand les feuilles vraies ne sont pas encore apparues. Partie 2: VIGS JOUR 1 <ol><li> Pour chaque expérience, Agrobacterium tumefaciens hébergeant pTRV1, pTRV2, pTRV2-PDS et gène cible pTRV2-hôtes sont cultivées sur des plaques d&#39;agar LB additionné de 50 pg / ml de kanamycine et 100 pg / mL de rifampicine. La kanamycine sélectionne pour la pTRV plasmide alors que la rifampicine fait pour l&#39;Agrobacterium. Incuber la plaque à 30 ° C pendant 2 jours. Désactivation du PDS causera les plantes à photobleach et est utilisé comme un contrôle d&#39;efficacité taire. En outre, les gènes à régulation négative doit être cloné dans un vecteur pTRV2. Il ya une passerelle compatible pTRV2 vecteur qui peut faciliter le clonage et est décrit par Liu et al. (2002). </li></ol> JOUR 3 <ol><li> Inoculer un 2 - 3 ml de culture liquide de LB avec les antibiotiques mentionnés ci-dessus pour chacune des souches. Incuber en secouant à 30 ° C pendant 16 - 18 heures et 200 tours par minute </li></ol> JOUR 4 <ol><li> Inoculer un 1: 25 de dilution de la culture primaire dans un média secondaire induction liquide (IM) la culture de messagerie instantanée avec la kanamycine, la rifampicine et 200 acétosyringone uM (tableaux 1, 2 et 3). Le acétosyringone est utilisé comme un inducteur des gènes vir d&#39;Agrobacterium qui sont requis pour le transfert de l&#39;ADN-T dans la plante 9 tandis que le MI imite l&#39;environnement de cette rencontre de pathogènes dans l&#39;apoplaste hôte. Incuber en secouant à 30 ° C pendant 20 - 24 heures et 200 tours par minute </li></ol> JOUR 5 <ol><li> Récolter les cellules par centrifugation pendant 10 minutes à 3000 x g. Remettre en suspension dans le même volume que la culture d&#39;origine a eu avec 10 mM MgCl2, 10 mM MES pH 5,5. Les cellules peuvent être vortexés doucement à les remettre en suspension. </li><li> Centrifugue les cellules pendant 10 minutes à 3000 x g. Remettre en suspension dans la moitié du volume de la culture d&#39;origine avec 10 mM MgCl2, 10 mM MES pH 5,5. </li><li> Préparer une suspension bactérienne avec une DO 600 de 0,3 pour chaque culture bactérienne. Ajouter acétosyringone à une concentration finale de 400 uM à la culture pTRV1. </li><li> Mélanger les cultures contenant de la pTRV1 et le pTRV2 (ou le pTRV2 contenant le gène d&#39;intérêt) dans un rapport 1 pour 1. Incluent également un contrôle pTRV2-PDS. S&#39;il vous plaît noter que la concentration finale est maintenant acétosyringone 200 uM et que chaque culture est à une DO 600 de 0,15. </li><li> Étiqueter les semis d&#39;être infiltré par le gène de se taire et la date de l&#39;expérience. </li><li> Percer un trou dans chaque feuille doit être infiltré avec une aiguille. Utiliser une seringue de 1 mL inutiles pour infiltrer la suspension bactérienne dans le semis. Pour la tomate, infiltrer deux cotylédons tandis que pour N. benthamiana, infiltrer les deux plus grandes feuilles vraies. Cinq ml de chaque mélange bactérien devrait être suffisant pour infiltrer 15 N. benthamiana et 25 plants de tomates. Évitez la contamination croisée en changeant de gants entre les infiltrations et par ne pas arroser les plantes avant le lendemain après l&#39;inoculation. </li><li> Les plantes sont maintenus à 20 - 22 ° C dans une chambre de croissance avec une longueur de journée de 16 heures et 50% HR pendant au moins 3 semaines et demie avant qu&#39;ils peuvent être utilisés pour les dosages. </li></ol> Partie 3: Les résultats représentatifs La figure 1 montre une expérience représentative avec N. benthamiana et des plants de tomates réduites au silence pour PDS. Plantes montrer le phénotype caractéristique de photoblanchiment observée dans les usines avec des quantités diminuées de caroténoïdes. Pour la PDS-taire des plantes témoins, commence photoblanchiment d&#39;être vu dès que 1 ½ semaines après l&#39;infiltration. <img src="/files/ftp_upload/1292/Figure 1A 1292.jpg" alt="Figure 1a" /><img src="/files/ftp_upload/1292/Figure 1B 1292.jpg" alt="figure 1b" /> Figure 1. Désactivation du gène de contrôle du PDS cause photoblanchiment de N. benthamiana (A) et la tomate (B) des plantes. Les photographies ont été prises trois semaines et demi après taire. Tableau 1. Préparation du milieu d&#39;induction (GI). <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 400 ml </td><td> H 2 O distillée </td></tr><tr><td> 4,88 g </td><td> MES (2 - (4 morpholino) éthane sulfonique) </td></tr><tr><td> 2,5 g </td><td> Le glucose </td></tr><tr><td> 0,12 g </td><td> NaH 2 PO 4 </td></tr></tbody></table> Porter à un volume final de 475 ml avec dH 2 O et ajuster le pH à 5,6. Autoclave. Après que le milieu est refroidi, ajouter 25 ml de sels AB 20X. Tableau 2. Préparation de sels AB. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 20 g </td><td> NH 4 Cl </td></tr><tr><td> 6 g </td><td> MgSO 4 · 7H 2 O </td></tr><tr><td> 3 g </td><td> KCl </td></tr><tr><td> 0,2 g </td><td> CaCl 2 </td></tr><tr><td> 0,05 g </td><td> FeSO 4 · 7H 2 O </td></tr></tbody></table> Porter à un volume final de 1 litre avec de l&#39;eau distillée. Autoclave. Soyez conscient que les sels AB précipiter sous forme d&#39;une poudre orange. Il suffit de les mélanger en remuant avant leur utilisation. Tableau 3. Préparation de 200 acétosyringone mM. S&#39;il vous plaît noter que acétosyringone doivent être préparés le jour où il sera utilisé. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 19,6 mg </td><td> Acétosyringone (3 &#39;, 5&#39;-diméthoxy-4&#39;-hydroxyacétophénone) </td></tr><tr><td> 500 ul </td><td> DMSO (diméthylsulfoxyde) </td></tr></tbody></table>

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	<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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+leaf+morphogenesis+by+microRNAs.">Control of leaf morphogenesis by microRNAs.</a> Nature. 425, 257-263 (2003).</li><li>Brigneti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+pathogenicity+determinants+are+suppressors+of+transgene+silencing+in+Nicotiana+benthamiana.">Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.</a> EMBO J. 17, 6739-6746 (1998).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant Journal. 31, 777-786 (2002).</li><li>MacFarlane, S. 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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+tumefaciens+and+plant+cell+interactions+and+activities+required+for+interkingdom+macromolecular+transfer.">Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer.</a> Annu. Rev. Cell Dev. Biol. 22, 101-127 (2006).</li><li>Rosebrock, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacterial+E3+ubiquitin+ligase+targets+a+host+protein+kinase+to+disrupt+plant+immunity.">A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.</a> Nature. 448, 370-374 (2007).</li><li>Miki, D., Itoh, R., Shimamoto, K. <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+of+single+and+multiple+members+in+a+gene+family+of+rice.">RNA silencing of single and multiple members in a gene family of rice.</a> Plant Physiol. 138, 1903-1913 (2005).</li><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=Use,+tolerance+and+avoidance+of+amplified+RNA+silencing+by+plants.">Use, tolerance and avoidance of amplified RNA silencing by plants.</a> Trends Plant Sci. 13, 317-328 (2008).</li><li>Ekengren, S. K., Liu, Y., Schiff, M., Kumar-Dinesh, S. P., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+MAPK+cascades,+NPR1,+and+TGA+transcription+factors+play+a+role+in+Pto-mediated+disease+resistance+in+tomato.">Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato.</a> Plant J. 36, 905-917 (2003).</li><li>Liu, E., Page, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cDNA+libraries+for+virus-induced+gene+silencing+(VIGS)+using+tobacco+rattle+virus.">Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus.</a> Plant. Methods. 4, 5-5 (2008).</li></ol>

Induite par le virus gene silencing est une méthode qui permet rapidement inversée écrans génétique. Il évite la génération de T-ADN ou le gène médié transposon knock-outs, qui ne sont disponibles que dans certaines plantes comme Arabidopsis et le maïs. Il contourne le processus fastidieux de transformation des plantes et permet le ciblage de gènes multiples dans le même temps, à condition qu&#39;ils aient une homologie suffisante 10 ou que les différentes séquences cibles d&#39;hôt...

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

L&#39;interférence ARN (ARNi) est un très spécifiques de silençage génique phénomène déclenché par une ARNdb. Ce mécanisme utilise taire deux grandes classes de régulateurs d&#39;ARN: les microARN, qui sont produites à partir non des gènes codant des protéines et des courts ARN interférents (siRNA). Les plantes utilisent l&#39;ARNi pour contrôler les transposons et d&#39;exercer un contrôle serré sur les processus de développement tels que la formation des organes de fleurs et de 2,3,4 développement des feuilles. Plantes également utiliser l&#39;ARNi pour se défendre contre l&#39;infection par des virus. En conséquence, de nombreux virus ont évolué suppresseurs de gènes taire pour permettre leur colonisation réussie de leur hôte 5. Induite par le virus gene silencing (VIGS) est une méthode qui prend avantage de l&#39;usine de l&#39;ARNi à médiation mécanisme de défense antivirale. Chez les plantes infectées par des virus non modifiés, le mécanisme est spécifiquement ciblées contre le génome viral. Cependant, avec des vecteurs viraux portant des séquences dérivées de gènes de l&#39;hôte, le processus peut en outre être ciblées contre les ARNm hôte correspondant. VIGS a été adapté à haut débit de génomique fonctionnelle chez les plantes en utilisant la plante pathogène Agrobacterium tumefaciens pour offrir, via son plasmide Ti, un virus recombinant portant la totalité ou une partie de la séquence du gène ciblé pour réduire au silence. La propagation du virus systémique et les plantes endogènes ARNi machines prendre soin du reste. dsRNA correspondant au gène cible sont produites et ensuite clivée par la ribonucléase Dicer en siRNA de 21 à 24 nucléotides de long. Ces siRNA finalement guider la RNA-Induced Silencing complexes (RISC) à dégrader la transcription cible 2. Différents vecteurs ont été utilisés dans VIGS et l&#39;un des plus fréquemment utilisée est basée sur le tabac hochet virus (TRV). VRT est un virus bipartite et, comme tels, A. différentes deux tumefaciens souches sont utilisées pour VIGS. On réalise pTRV1, qui code les fonctions de réplication virale et le mouvement tandis que l&#39;autre, pTRV2, ports la protéine d&#39;enveloppe et de la séquence utilisée pour VIGS 6,7. L&#39;inoculation de Nicotiana benthamiana et plants de tomates avec un mélange de souches des résultats aussi bien dans le silençage génique. Désactivation de l&#39;endogènes phytoène désaturase (PDS) du gène, ce qui provoque photoblanchiment, est utilisé comme un contrôle d&#39;efficacité VIGS. Il faut noter, cependant, que l&#39;inhibition de la tomate est généralement moins efficace que dans N. benthamiana. L&#39;abondance de transcription d&#39;ARN du gène d&#39;intérêt doit toujours être mesuré afin de s&#39;assurer que le gène cible a été efficacement régulé à la baisse. Néanmoins, les séquences de gènes hétérologues de N. benthamiana peut être utilisé pour réduire au silence leurs orthologues chez la tomate et 8 vice-versa.

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

Description d&#39;une induite par le virus gene silencing (VIGS) méthode pour le knock-down de l&#39;expression génique dans le<em&gt; Nicotiana benthamiana</em&gt; Et la tomate.

Induite par le virus Gene Silencing (VIGS) dans Nicotiana benthamiana Et aux tomates

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

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Biology

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

52.4K vues.  Cornell University. Description d'une induite par le virus gene silencing (VIGS) méthode pour le knock-down de l'expression génique dans le Nicotiana benthamiana Et la tomate.

Vidéo: 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.

Direct imagerie calcique cellulaire Combiné avec Gene Silencing siARN Mediated Identifie Ca 2 + Canaux de fuite dans la membrane du RE et de leurs mécanismes de régulation

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

Recherche

Biologie

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.

Induction et les essais de l&#39;hypoxie en culture cellulaire

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.

Agrobacterium induite par le virus Assay Gene Silencing En Coton

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.

Isolement du ribosome Bound polypeptides naissants<em&gt; In vitro</em&gt; Pour identifier les sites de pause de translation le long d&#39;ARNm

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.

Utilisation de la séquence d&#39;arrêt SecM comme un outil pour isoler les polypeptides Ribosome Bound

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.

Agroinfiltration et PVX Agroinfection dans la pomme de terre et Nicotiana benthamiana

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.

Optimisation et l&#39;utilisation des<em&gt; Agrobacterium</em&gt; Médiée par la production de protéines transitoire dans<em&gt; Nicotiana</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.

Chitosane / ARN interférant nanoparticules Mediated Gene Silencing dans la maladie vectorielle larves de moustiques

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.

Meiotic évaluation de la broche dans la souris ovocytes par silençage siRNA médiation

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.

Analyse de l’imagerie de la complémentation de luciférase dans les feuilles de Nicotiana benthamiana pour transitoirement déterminant la dynamique Interaction protéine-protéine

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