Nous sommes reconnaissants aux Drs. SP Dinesh Kumar et Yule-Liu pour TRV-VIGS vecteurs, et les Drs. Chuck Kenerley, Terry Wheeler, Jim Starr et Bayer CropScience pour fournir des semences de coton. Ce travail a été financé par la NSF à LS et les NIH à PH

1. Pousser les semis de coton <ol><li> Semer les graines du coton upland (Gossypium hirsutum) variété Fibermax 832, Phytogen 425RF, Phytogen 480WR et Deltapine 90 dans des pots (7 cm de diamètre) contenant Metro Mix 700 (SunGR, Beavile, WA). </li><li> Gardez les pots dans un plateau recouvert d&#39;un dôme en plastique à 23 ° C, 120 uE m -2 s -1 lumière, avec une photopériode 12 heures heure de lumière/12 sombre dans une chambre de croissance. </li><li> Retirez le dôme lorsque deux cotylédons ont émergé. </li><li> Environ deux semaines plus tard, l&#39;utilisation des plantes avec deux cotylédons sont entièrement développés pour le dosage VIGS. A ce stade, les vraies feuilles n&#39;ont pas encore émergé (figure 1). </li></ol> 2. Construire vecteur VIGS transportant GrCLA1 ​​génique <ol><li> Amplifier le alterados Cloroplastos 1 gène de coton (GrCLA1) par PCR avec des amorces GrCLA1-F, 5&#39;-GCCCTTTGTGCATCTTC-3 &#39;et GrCLA1-R, 5&#39;-CTCTAGGGGCATTGAAG-3&#39; à partir d&#39;une bibliothèque d&#39;ADNc de G. raimondii tissus foliaires. </li><li> Recueil des produits de PCR de GrCLA1 ​​avec EcoRI et Kpnl et insérez-le dans pYL156 (pTRV-RNA2) vecteur par ligature. </li><li> Ecran clones sur des plaques d&#39;agar LB contenant 50 pg / ml de kanamycine. Vérifiez les constructions par digestion d&#39;enzymes de restriction et le séquençage. </li><li> Transformer le plasmide dans Agrobacterium tumefaciens GV3101 par électroporation et de récupérer dans un milieu liquide LB à 28 ° C. Sélectionnez les transformants sur plaques avec le LB + kanamycine (50 pg / ml) + gentamicine (25 ug / ml). Les bactéries peuvent être stockés dans le glycérol 25% à -80 ° C pour une utilisation à long terme. </li></ol> 3. Effectuer VIGS l&#39;inoculation <ol><li> Trois jours avant l&#39;inoculation stocks strie, glycérol congelés d&#39;Agrobacterium tumefaciens portant pYL192 (VRT): ARN1, pYL156 (VRT): RNA2 (vecteur seul) et pYL156 (VRT): RNA2-GrCLA1 ​​sur les plaques d&#39;agar LB contenant 50 ug / ml de kanamycine et 25 pg / ml de gentamycine. Incuber les plaques à 28 ° C pendant 24 heures. </li><li> Deux jours avant l&#39;infiltration VIGS, choisissez une seule colonie pour chaque construction à partir des plaques ci-dessus et inoculer dans 5 ml de milieu LB additionné de 50 pg / ml de kanamycine et 25 pg / ml de gentamycine; Cultiver la culture bactérienne à 28 ° C pour la nuit dans un tambour à rouleaux à une vitesse de 50 rpm. </li><li> Transférer la culture au-dessus d&#39;un flacon avec 50 ml de milieu LB additionné de 50 pg / ml de kanamycine et 25 pg / ml de gentamycine, majoré de 10 mM de MES (2 - (4 morpholino) éthane sulfonique) et 20 acétosyringone uM. Cultiver la culture à 28 ° C pour la nuit dans un shaker avec une vitesse de 50 rpm. </li><li> Le jour suivant, ralentit les cellules agro-bactérienne à 4000 rpm pendant 5 minutes; remettre en suspension de la culture dans le tampon d&#39;infiltration contenant 10 mM MgCl2, 10 mM de MES et 200 acétosyringone uM. Réglez la DO 600 de la culture à 1,5. </li><li> Laisser la culture sur le banc à la température ambiante pendant 3 heures. </li><li> Avant l&#39;infiltration d&#39;agrobactéries, de punch en dessous des cotylédons des plants de coton avec une aiguille 25 G sans percer à travers les cotylédons. Un ou deux trous (dépend de la douceur des tissus) ont été perforées sur chaque section du cotylédon (figure 2). </li><li> Mélanger la suspension de la culture d&#39;agrobactéries pYL192 (VRT): ARN1 et pYL156 (VRT): RNA2 ou pYL156 (VRT): RNA2-GrCLA1 ​​dans un rapport 1:1; main d&#39;infiltrer le mélange du dessous de cotylédons à travers les sites utilisant un blessant une mL sans aiguille de seringue (figure 3). </li><li> Couvrez les plantes avec un dôme de plastique et laisser les plantes infiltrées à température ambiante sous condition de faible lumière pour la nuit. </li><li> Transfert plantes pour une chambre de croissance avec la température de 23 ° C, 120 uE m -2 s -1 lumière avec un cycle de lumière heures / 12 12 heures d&#39;obscurité. </li></ol> Remarque: VIGS fonctionne lorsque les plantes sont sous soit 23 ° C ou en serre (25-28 ° C). Lorsque la température est relativement basse (23 à 25 ° C), il est plus facile pour l&#39;inoculation et le phénotype est plus uniforme par rapport supérieur (28-30 ° C) ou la température fluctue. Couvrant les plantes avec un dôme permet également VIGS plus efficace. <ol start="10"><li> Examinez le phénotype taire à 7 ~ 8 jours après l&#39;infiltration. Les vraies feuilles de plantes réduites au silence par pYL156 (VRT): RNA2-GrCLA1 ​​commencé à afficher le phénotype albinos (figure 4). Les plantes infiltrées par la réalisation agrobactéries pYL156 (VRT): RNA2 servir de contrôle. </li><li> Vérifier l&#39;efficacité inactivation des gènes en examinant le niveau d&#39;expression de gènes endogènes en utilisant la transcription inverse de réaction en chaîne polymérase (RT-PCR) avec l&#39;ARN isolé de contrôle et de plants de coton au silence. </li></ol> Note: Les plantes VIGS-ED doivent être maintenus sous des conditions contenues quarantaine et les plantes doivent être bien à l&#39;autoclave et éliminés après les tests que VRT a un large spectre d&#39;hôte et est un agent pathogène à déclaration obligatoire. 4.Les résultats représentatifs: Environ deux semaines après la main-imprégnation avec le mélange d&#39;agrobactéries, le phénotype albinos causés par GrCLA1 ​​taire a été clairement observé sur les vraies feuilles. L&#39;efficacité taire atteint presque 100% dans de multiples expériences avec plus de 50 plantes. Les plantes présentent un phénotype taire uniformément répartie et très forte sur les albinos, nouvellement développés laisse avec une mosaïque sur les feuilles âgées (figure 4). <img src="/files/ftp_upload/2938/2938fig1.jpg" alt="Figure 1" /> Figure 1. Un plant de coton d&#39;environ deux semaines avec deux vieux stade de cotylédons sont entièrement développés utilisé pour l&#39;infiltration agrobactéries. <img src="/files/ftp_upload/2938/2938fig2.jpg" alt="Figure 2" /> Figure 2. Poinçonnage des trous minuscules sur le dessous de cotylédons des plantes de coton à l&#39;aide d&#39;une aiguille 25 G pour faciliter l&#39;infiltration agrobactéries. <img src="/files/ftp_upload/2938/2938fig3.jpg" alt="Figure 3" /> Figure 3. Infiltration de la main d&#39;un mélange d&#39;agrobactéries dans les cotylédons de coton par les sites en blessant l&#39;aide d&#39;une seringue sans aiguille <img src="/files/ftp_upload/2938/2938fig4.jpg" alt="Figure 4" /> Figure 4. Phénotype albinos est apparu sur VIGS taire les plants de coton. Quatre cultivars sont présentés. A) Fibermax 832; B) Phytogen 480WR; C) Phytogen 425RF; D) Deltapine 90.

<ol>
	<li>Chen, Z. J., Scheffler, B. E., Dennis, E., Triplett, B. A., Zhang, T., Guo, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+sequencing+cotton+(Gossypium)+genomes.">Toward sequencing cotton (Gossypium) genomes.</a> Plant Physiol. 145, 1303-1310 (2007).</li><li>Dinesh-Kumar, S. P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y. <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.">Virus-induced gene silencing.</a> Methods Mol Biol. 236, 287-294 (2003).</li><li>Hamilton, A. J., Baulcombe, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+species+of+small+antisense+RNA+in+posttranscriptional+gene+silencing+in+plants.">A species of small antisense RNA in posttranscriptional gene silencing in plants.</a> Science. 286, 950-952 (1999).</li><li>Burch-Smith, T. M., Anderson, J. C., Martin, G. B., 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=Applications+and+advantages+of+virus-induced+gene+silencing+for+gene+function+studies+in+plants.">Applications and advantages of virus-induced gene silencing for gene function studies in plants.</a> Plant J. 39, 734-746 (2004).</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 J. 31, 777-786 (2002).</li><li>Estevez, J. M., Cantero, A., Romero, C., Kawaide, H., Jimenez, L. F., Kuzuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+expression+of+CLA1,+a+gene+that+encodes+the+1-deoxyxylulose+5-phosphate+synthase+of+the+2-C-methyl-D-erythritol-4-phosphate+pathway+in+Arabidopsis.">Analysis of the expression of CLA1, a gene that encodes the 1-deoxyxylulose 5-phosphate synthase of the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis.</a> Plant Physiol. 124, 95-104 (2000).</li><li>Mandel, M. A., Feldmann, K. A., Herrera-Estrella, L., Rocha-Sosa, M., Leon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLA1,+a+novel+gene+required+for+chloroplast+development,+is+highly+conserved+in+evolution.">CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.</a> Plant J. 9, 649-658 (1996).</li><li>Gao, X., Wheeler, T., Li, Z., Kenerley, C., He, P., Shan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+GhNDR1+and+GhMKK2+compromised+cotton+resistance+to+Verticillium+wilt.">Silencing GhNDR1 and GhMKK2 compromised cotton resistance to Verticillium wilt.</a> Plant J. 66, 293-305 (2011).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

VIGS a été prouvé être un outil puissant dans l&#39;analyse génomique fonctionnelle par transitoirement renversant l&#39;expression de gènes endogènes. Dans cette étude, nous avons développé une VIGS Agrobacterium en utilisant un vecteur binaire basée VRT. Le coton CLA1 (GrCLA1) gène a été développé comme un repère visuel pour surveiller l&#39;efficacité taire. Nous avons toujours obtenu 100% d&#39;efficacité silençage génique, a démontré par le phénotype albinos apparaissant sur les feu...

<table border="1"><tbody><tr><th> Nom des réactifs et des équipements </th><th> Société </th><th> Catalogue / numéro de série </th><th> Commentaires </th></tr><tr><td> Rouleau à tambour </td><td> Glas-Col, LLC. </td><td> 099A TC108 </td><td> Agro-bactérie de la culture </td></tr><tr><td> Incubateur </td><td> Sheldon Manufacturing, Inc </td><td> 01046209 </td><td> Agro-bactérie de la culture </td></tr><tr><td> UV / Vis spectrophotomètre </td><td> Beckman Coulter </td><td> Modèle: DU530 </td><td> Mesure OD </td></tr><tr><td> Gene Pulser </td><td> BioRad </td><td> Modèle: 1652076; de série: 154BR3880 </td><td> L&#39;électroporation </td></tr><tr><td> Pulser contrôleur </td><td> BioRad </td><td> Modèle: 1652098; de série: 232BR4833 </td><td> L&#39;électroporation </td></tr><tr><td> Micropulser électroporation cuvette </td><td> BioRad </td><td> 165-2081 </td><td> L&#39;électroporation </td></tr><tr><td> Seringue de 1 mL </td><td> BD Biosciences </td><td> 30962 </td><td> L&#39;inoculation de l&#39;agro-bactérie </td></tr><tr><td> Metro Mix 700 </td><td> SUNGR </td><td> SKU # 553001 </td><td> Plantule croissance </td></tr><tr><td> Terra Cotta pot </td><td> TOPlastics </td><td> GPS 3001B2 </td><td> Plantule croissance </td></tr><tr><td> Monohydraté MES </td><td> USB </td><td> 18886 </td><td> Mémoire tampon d&#39;infiltration </td></tr><tr><td> Acétosyringone </td><td> Sigma </td><td> D134406 </td><td> Mémoire tampon d&#39;infiltration </td></tr></tbody></table>

agrobacterium-mediated virus-induced gene silencing assay in cotton

Cotonnier (Gossypium hirsutum) est l&#39;une des cultures les plus importantes dans le monde entier. Des efforts considérables ont été réalisés sur l&#39;amélioration moléculaire des variétés nouvelles. L&#39;analyse génétique à grande échelle fonctionnelle de coton a été en retard sur la plupart des espèces végétales modernes, probablement en raison de sa grande taille de la duplication du génome, le gène et la polyploïdie, cycle de croissance long et récalcitrant à la transformation génétique 1. Pour faciliter l&#39;étude fonctionnelle à haut débit génétique / génomique dans le coton, nous essayons de développer des tests rapides et efficaces transitoires afin d&#39;évaluer les fonctions des gènes de coton. Virus-Induced Gene Silencing (VIGS) est une technique puissante qui a été développé sur la base de l&#39;hôte inactivation génique post-transcriptionnelle (PTGS) pour réprimer la prolifération virale 2,3. Agrobacterium VIGS a été appliquée avec succès dans un large éventail d&#39;espèces dicotylédones telles que des Solanacées, Arabidopsis et des espèces de légumineuses, et des espèces monocotylédones dont l&#39;orge, le blé et le maïs, pour diverses études génomiques fonctionnelles 3,4. Comme cette approche rapide et efficace évite la transformation des plantes et surmonte redondance fonctionnelle, il est particulièrement attrayant et adapté pour l&#39;étude de génomique fonctionnelle chez les espèces de cultures comme le coton ne se prêtent pas à la transformation. Dans cette étude, nous présentons le protocole détaillé du système d&#39;Agrobacterium VIGS en coton. Parmi les vecteurs viraux plusieurs VIGS, le virus du rattle (VRT) envahit un large éventail d&#39;hôtes et est capable de se propager à travers vigoureusement la plante entière encore produire des symptômes bénins sur le hosts5. Pour surveiller l&#39;efficacité taire, GrCLA1, un gène homologue de Arabidopsis Cloroplastos alterados une gène (AtCLA1) en coton, a été cloné et inséré dans le code binaire VIGS vecteur pYL156. CLA1 gène est impliqué dans le développement des chloroplastes 6, et les études précédentes ont montré que perte de fonction de AtCLA1 abouti à un phénotype albinos sur les vraies feuilles 7, offrant un excellent marqueur visuel pour faire taire l&#39;efficacité. À environ deux semaines après l&#39;infiltration d&#39;Agrobacterium, le phénotype albinos ont commencé à apparaître sur les feuilles vraies, avec une efficacité de 100% en silence toutes les expériences répétées. La réduction au silence l&#39;expression des gènes endogènes a également été confirmée par analyse RT-PCR. De manière significative, pourrait faire taire puissamment se produisent dans tous les cultivars que nous avons testés, y compris les diverses variétés cultivées commercialement au Texas. Cette rapide et efficace au moyen d&#39;Agrobacterium test VIGS fournit un outil très puissant pour rapide et à grande échelle d&#39;analyse des fonctions des gènes au niveau de l&#39;échelle du génome du coton.

Watch this Scientific Journal Video about Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton at JoVE.com

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Nous présentons le protocole détaillé pour Agrobacterium induite par le virus du gène taire (VIGS) de dosage dans le coton. Le virus du rattle (VRT) vecteurs dérivés VIGS ont été déployés pour inciter RNA silencing de coton GrCLA1, alterados Cloroplastos une gène. Le phénotype albinos causés par faire taire GrCLA1 ​​a été observée au stade plantule dans les 2 semaines après l&#39;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 ...

agrobacterium-mediated-virus-induced-gene-silencing-assay-in-cotton

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24.2K vues.  Texas A&M University. Nous présentons le protocole détaillé pour Agrobacterium induite par le virus du gène taire (VIGS) de dosage dans le coton. Le virus du rattle (VRT) vecteurs dérivés VIGS ont été déployés pour inciter RNA silencing de coton GrCLA1, alterados Cloroplastos une gène. Le phénotype albinos causés par faire taire GrCLA1 ​​a été observée au stade plantule dans les 2 semaines après l'inoculation.

Vidéo: Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

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

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

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

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

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 ...

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Biologie

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 ...

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 ...

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 ...

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 ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Agrobacterium-Transformation d’embryon immature médié des lignées consanguines de maïs récalcitrants utilisant des gènes morphogènes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...