Nous remercions James Clark pour fournir des figures d&#39;animaux à différents points dans le temps de développement. C. souches elegans dans cette étude ont été obtenues à partir du Centre de Caenorhabditis Genetics, qui est soutenu par le Centre National de Ressources NIH Research (NCRR). Ce travail a été soutenu par CIRG 1817 à partir de CUNY à JL et CDD, et par le NIH 1R15GM073678-01-01 et 1R15GM097692 à la CDD Nous remercions le Dr William J Rice, le Dr Nathalia Holtzman, et Mélissa Silvestrini pour les commentaires sur le manuscrit. Ce travail a été réalisé dans l&#39;accomplissement partiel des exigences d&#39;un doctorat de la Graduate Center de la City University of New York (S. Xiong).

1. Préparation des plaques d&#39;alimentation ARNi séquence porteuse gène cible <ol><li> Si vous utilisez les bibliothèques disponibles dans le commerce ARNi (par exemple à partir de LifeSciences Source BioScience), passez à l&#39;étape 1.4. Alternativement, cloner des séquences de gènes cibles dans le vecteur L4440, un ver couramment utilisé ARNi plasmide 8, par protocole de clonage standard. </li><li> Transformer le plasmide recombinant dans la souche bactérienne HT115 (DE3), la culture sur les plaques d&#39;agar LB avec 25 ug / ml de carbénicilline et 12,5 pg / ml tétracycline, et de choisir un seul clone pour une utilisation future. </li><li> Pendant ce temps, transformer le L4440 vecteur vide dans la souche bactérienne HT115 pour l&#39;utiliser comme une commande pour les expériences. </li><li> Culture fois recombinants et vide L4440-porteurs de bactéries dans 1 ml de bouillon LB avec 100 pg / ml d&#39;ampicilline pendant la nuit à 37 ° C. Tétracycline n&#39;est pas ajouté en raison du fait qu&#39;elle réduit l&#39;efficacité ARNi. </li><li> Ajouter un autre bouillon de 5 LB ml avec 100 pg / ml d&#39;ampicilline dans la nuit culture, incuber pendant une autre heure 6.4 à 37 ° C. </li><li> Seed 0,5 ml recombinant ou vide L4440-portant culture bactérienne sur les plaques à vis sans fin ARNi. Étiquetez toutes les plaques avec le nom de clone. </li><li> Marquer les plaques et incuber pendant une nuit à 37 ° C pour augmenter tapis bactérien, ce sont les plaques d&#39;ARNi avec ou sans séquence de gène cible. Au moins 2 plaques doivent être préparés pour chaque condition. </li></ol> 2. Worms Culture sur les plaques d&#39;alimentation ARNi <ol><li> Flamme stériliser la pointe d&#39;un pick ver fil de platine. Utilisez le ver choisir de prendre 6-10 quatrième stade larvaire (L4) hermaphrodites à chaque plaque ARNi qui contient soit L4440 recombinant ou vides, les animaux utilisent les bactéries comme source de nourriture. </li><li> Laissez hermaphrodites croître à 20 ° C (une des conditions de culture standard pour C. elegans) pendant la nuit, ils deviennent de jeunes adultes le lendemain. </li><li> Transfert 6-10 jeunes adultes dans une nouvelle plaque ARNi en conséquence étiquetés et prêts. </li><li> Laissez leadultes pondent des œufs pour 4-6 heures, puis supprimez tous les adultes à partir de la plaque; cette étape consiste à synchroniser la descendance. Une fois que les mères sont enlevées, commencez à compter le temps. C&#39;est le temps zéro. </li><li> Incuber les plaques à 20 ° C pour permettre aux animaux grandissent au stade de développement spécifique, dans ce protocole, nous choisissons les jeunes adultes pour l&#39;analyse phénotypique. Pour passes rabattues qui se développent à un rythme normal, 72 heures d&#39;incubation est suffisante pour que les animaux deviennent de jeunes adultes. Cependant, différents gènes influent sur le développement des animaux différemment. Si RNAi entraîne les animaux à se développer à un rythme différent, les jeunes adultes peuvent être identifiés comme étant ceux h pas plus de 24 L4 passé avec le développement vulvaire complété et 2-6 embryons dans l&#39;utérus (si fertile). Pour identifier d&#39;autres stades de développement, l&#39;enquêteur doit utiliser le développement des gonades et de la vulve comme un guide. </li></ol> 3. Phénotypes note d&#39;état corporel Taille de l&#39;ARNi traité de Worms <ol><li> Placez deux couches de rubans d&#39;étiquettes colorées sur une lame de verre. Assurez-deux deslames de verre soi. Puis, placer une lame de verre à nouveau entre les deux lames de ruban. Faire fondre agarose à 2% dans l&#39;eau, appliquer une goutte au centre de la lame de verre nouveau, puis appuyez sur la lame de verre deuxième sur le dessus pour faire une fine couche de 2% d&#39;agarose comme décrit précédemment 9. </li><li> Une fois que l&#39;agarose est solidifié, retirer la lame de verre haut, la lame d&#39;agarose pad est prêt à charger vers. Glissière étiquette avec le nom de clone. </li><li> Ajouter 10 ul 25 mm 3 NaN pour le pad agarose; prendre 30-40 individus dans la solution de NaN3 de les immobiliser, puis mettre la lamelle sur le dessus; répétez l&#39;opération pour les deux recombinante et vide L4440-portant RNAi chez les animaux traités. </li><li> L&#39;image des vers sous loupe binoculaire à l&#39;aide objectif 2.5x, ici nous utilisons Leica appareil photo numérique avec l&#39;appui Qcapture logiciel. </li><li> Pour l&#39;étalonnage, d&#39;abord capturer une image d&#39;un souverain micromètre standard. Ensuite, ouvrez l&#39;image dans l&#39;image-Pro, Cliquez sur &quot;mesure&quot; et choisissez &quot;étalonnage&quot;, puis choisissez«Assistant de calibrage spatial». Avec «calibrer l&#39;image active&quot; est sélectionné, cliquez sur &quot;suivant&quot;. Saisissez le nom de l&#39;étalonnage et de veiller à ce que les unités de référence spatiale sont mis à micromètre. Ensuite, cochez la case &quot;créer un étalonnage de référence&quot; et cliquez sur &quot;Suivant&quot;. Cliquez sur &quot;ligne de référence tirage au sort.&quot; Un bar échelle de référence apparaît. Repositionner la barre d&#39;échelle pour faire correspondre les extrémités du micromètre, puis d&#39;indiquer que la référence indique 1.000 unités. Cliquez sur &quot;OK&quot;, &quot;Suivant&quot; et &quot;Terminer&quot;. </li><li> Une fois que le logiciel est calibré, ouvrez une image ver. Ensuite, sélectionnez dans la &quot;mesure&quot;, sélectionnez &quot;calibration&quot; puis cliquez sur &quot;select spatiale&quot;. Dans le menu déroulant, sélectionnez le nom du fichier créé pour l&#39;étalonnage du micromètre. Puis, sous la &quot;mesure&quot;, sélectionnez &quot;mesures&quot;. Dans la fenêtre qui s&#39;ouvre, sélectionnez l&#39;outil à dessiner et tracer une ligne passant par le centre du corps de l&#39;animal, de la tête à la queue avec la souris d&#39;ordinateur (Figure 1). La longueur sera signalée dans la fenêtre. Maintenantvous avez deux groupes de données: la longueur du corps des animaux traités par ARNi des gènes cibles et les animaux témoins traités avec vide L4440 vecteur. </li><li> Exportez les mesures de longueur dans un fichier Microsoft Excel ou d&#39;autres logiciels de statistique appropriée. Analyser les données en calculant la moyenne et l&#39;écart-type pour chaque échantillon. Puis comparer les échantillons à l&#39;aide de Student t-test. </li></ol>

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	<li>Melnyk, C. W., Molnar, A., 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=Intercellular+and+systemic+movement+of+RNA+silencing+signals.">Intercellular and systemic movement of RNA silencing signals.</a> Embo J. 30, 3553-3563 (2011).</li><li>Wall, N. R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+RNA:+can+RNA+interference+be+exploited+for+therapy.">Small RNA: can RNA interference be exploited for therapy.</a> Lancet. 362, 1401-1403 (2003).</li><li>Kalantidis, K., Schumacher, H. T., Alexiadis, T., Helm, J. M. <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+movement+in+plants.">RNA silencing movement in plants.</a> Biol. Cell. 100, 13-26 (2008).</li><li>Shim, M. S., Kwon, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+targeted+delivery+of+siRNA+in+vivo.">Efficient and targeted delivery of siRNA in vivo.</a> Febs. J. 277, 4814-4827 (2010).</li><li>Amarzguioui, M., Rossi, J. J., Kim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+for+chemically+synthesized+siRNA+and+vector-mediated+RNAi.">Approaches for chemically synthesized siRNA and vector-mediated RNAi.</a> FEBS Lett. 579, 5974-5981 (2005).</li><li>Kamath, R. S., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screening+in+Caenorhabditis+elegans.">Genome-wide RNAi screening in Caenorhabditis elegans.</a> Methods. 30, 313-321 (2003).</li><li>Savage-Dunn, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screen+for+small+body+size+mutants+in+C.+elegans+reveals+many+TGFb+pathway+components.">Genetic screen for small body size mutants in C. elegans reveals many TGFb pathway components.</a> Genesis. 35, 239-247 (2003).</li><li>Timmons, L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+interference+by+ingested+dsRNA.">Specific interference by ingested dsRNA.</a> Nature. 395, 854 (1998).</li><li>Sulston, J. E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embryonic+cell+lineages+of+the+nematode,+Caenorhabditis+elegans.">Post-embryonic cell lineages of the nematode, Caenorhabditis elegans.</a> Dev. Biol. 56, 110-156 (1977).</li><li>Wang, J., Tokarz, R., Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+of+TGF&#946;+signal+transducers+in+the+hypodermis+regulates+body+size+in+C.+elegans.">The expression of TGF&#946; signal transducers in the hypodermis regulates body size in C. elegans.</a> Development. 129, 4989-4998 (2002).</li><li>Liang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+schnurri+homolog+sma-9+mediates+stage-+and+cell+type-specific+responses+to+DBL-1+BMP-related+signaling.">The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling.</a> Development. 130, 6453-6464 (2003).</li><li>Savage-Dunn, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;+signaling.">TGF-&#946; signaling.</a> WormBook. , 1-12 (2005).</li><li>Simmer, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+the+putative+RNA-directed+RNA+polymerase+RRF-3+makes+C.+elegans+hypersensitive+to+RNAi.">Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi.</a> Curr. Biol. 12, 1317-1319 (2002).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+misexpression+of+germline+P+granules+and+enhanced+RNA+interference+in+retinoblastoma+pathway+mutants.">Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.</a> Nature. 436, 593-597 (2005).</li><li>De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evo-devo:+variations+on+ancestral+themes.">Evo-devo: variations on ancestral themes.</a> Cell. 132, 185-195 (2008).</li><li>Timmons, L., Court, D. L., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ingestion+of+bacterially+expressed+dsRNAs+can+produce+specific+and+potent+genetic+interference+in+Caenorhabditis+elegans.">Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans.</a> Gene. 263, 103-112 (2001).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Dans ce protocole, nous décrivons notre méthode pour l&#39;identification des mutants défectueux de la taille du corps C. elegans par l&#39;alimentation ARNi. Cette méthode est applicable à l&#39;identification des composants de signalisation du TGF-β. Depuis ces composants sont hautement conservées à travers l&#39;évolution 15, ces écrans sont pertinents pour élucider les mécanismes moléculaires de la signalisation du TGF-β dans tous les métazoaires. Une considération importante dans...

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Plaques à vis sans fin ARNi 17,7 g Mélanger moyen Worm 60 g Agar Ajouter H 2 O à 3 litres. Autoclave. Ajouter 3 ml d&#39;IPTG 1M (stérile) et 3 ml 25 mg / ml carbénicilline (stérile), puis verser dans des boîtes de 60 mm de Pétri. Plaques à vis sans fin 17,7 g Mélanger moyen Worm 0,6 g de sulfate de streptomycine 60 g Agar Ajouter H 2 O à 3 litres. Autoclave. Verser dans 60 boîtes de Pétri mm. Mix Moyen Worm 55 g de Tris-Cl 24 g de Tris-OH 310 g Bacto Peptone 800 mg de cholestérol 200 g de NaCl Mélanger soigneusement et veillez à éviter les morceaux, ce mélange en poudre peut être conservée pendant de nombreux mois avant de les utiliser. Agarose à 2% 0,5 g d&#39;agarose Annonced 25 ml dH 2 O. La chaleur au micro-ondes jusqu&#39;à dissolution. 1M isopropyl β-D-1-thiogalactopyranoside (IPTG) 2 g IPTG Dissoudre in10 ml dH 2 O 1M NaN 3 6,5 g de NaN3 Ajouter dH 2 O à 100 ml 25 mM NaN 3 2,5 ml 1M NaN 3 Ajouter dH 2 O à 100 ml Caenorhabditis elegans de Caenorhabditis du Genetics Center L4440 plasmide (porte résistance à l&#39;ampicilline gène) Souche de bactéries HT115 (DE3) (contient IPTG polymérase T7 inductible; déficient pour la RNAse III gène (dsRNase) qui porte également la tétracycline gène de résistance) 16 60 mm boîtes de Pétri 100 mm boîtes de Pétri 14 ml à fond rond tubes de culture Falcon lames de verre lamelles de verre 1-20 pipette ul 20-200 ul pipette 200-1000 ulpipette 1-200 embouts de pipette ul 200-1000 embouts de pipette ul Tubes Eppendorf de 1,5 ml choix ver fil de platine </td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

Double-brin d&#39;ARN médiée par interférence (ARNi) est une stratégie efficace pour faire tomber l&#39;expression du gène cible 1-3. Il a été appliqué à de nombreux systèmes modèles, y compris les plantes, les invertébrés et les vertébrés. Il existe différentes méthodes pour atteindre 4,5 ARNi in vivo. Par exemple, le gène cible peut être transformé en un vecteur d&#39;ARNi, puis de façon permanente ou transitoire transformé dans des lignées cellulaires ou des cellules primaires pour obtenir des effets inactivation génique; bien synthétisées oligonucléotides double brin de gènes cibles spécifiques (ARNi oligos) peut être transitoirement dans des lignées cellulaires transformées ou des cellules primaires pour réduire au silence des gènes cibles, ou double-brin synthétisé les molécules d&#39;ARN peuvent être micro-injectées dans l&#39;organisme. Depuis le nématode C. elegans utilise des bactéries comme source de nourriture, nourrir les animaux avec des bactéries exprimant double-brin d&#39;ARN contre les gènes cibles fournit une stratégie viable 6. Nous présentons ici une alimentation ARNiméthode pour marquer phénotype taille du corps. Taille du corps chez C. elegans est régulée principalement par le TGF-β - ligand comme DBL-1, si ce test est approprié pour l&#39;identification du TGF-β composants de signalisation 7. Nous avons utilisé différentes souches dont deux souches ARNi hypersensibles à répéter les expériences d&#39;alimentation ARNi. Nos résultats ont montré que far-3 souche nous a donné le meilleur phénotype attendu ARNi. La méthode est facile à réaliser, reproductible et facilement quantifiables. En outre, notre protocole minimise l&#39;utilisation de l&#39;équipement spécialisé, il est donc approprié pour les petits laboratoires ou les établissements majoritairement de premier cycle.

Dans notre recherche, nous nous concentrons sur la réglementation taille du corps par la voie DBL-1/TGF-β. La perte de fonction des résultats voie DBL-1 dans une petite taille, y compris une longueur plus courte du corps par rapport à des animaux de type sauvage 7,10,11. Ainsi, écrans pour C. elegans mutants taille du corps sont capables d&#39;identifier les composants de signalisation TGF-β et de modificateurs. DBL-1 signalisation est médiée par une conservée TGF-β voie de transduction de ...

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Nous montrons comment utiliser la technique d&#39;alimentation ARNi pour abattre gènes cibles et le phénotype taille score de corps<em&gt; C. elegans</em&gt;. Cette méthode pourrait être utilisée pour un écran à grande échelle pour identifier les éventuels composants génétiques d&#39;intérêt, tels que ceux qui sont impliqués dans la régulation de la taille du corps par la signalisation DBL-1/TGF-β.

En utilisant l&#39;ARN médiée par la stratégie d&#39;alimentation interférences à l&#39;écran pour des gènes impliqués dans le règlement de la taille du corps du nématode<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

using-rna-mediated-interference-feeding-strategy-to-screen-for-genes

Research

JoVE Journal

Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

12.2K vues.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). Nous montrons comment utiliser la technique d'alimentation ARNi pour abattre gènes cibles et le phénotype taille score de corps C. elegans. Cette méthode pourrait être utilisée pour un écran à grande échelle pour identifier les éventuels composants génétiques d'intérêt, tels que ceux qui sont impliqués dans la régulation de la taille du corps par la signalisation DBL-1/TGF-β.

Vidéo: Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Application d&#39;une Assay C. elegans dégénérescence des neurones dopaminergiques pour la validation du potentiel de la maladie de Parkinson gènes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Recherche

Biologie

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Anticorps coloration dans<em&gt; C. Elegans</em&gt; Utilisation de «gel-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

À grande échelle Gene Knockdown dans<em&gt; C. elegans</em&gt; Utilisation ARNdb alimentation bibliothèques pour générer des phénotypes robustes perte de fonction

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Méthodes d&#39;évaluation de Subcellular compartiments de muscle en<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

Suivi et quantification des processus de développement des<em&gt; C. elegans</em&gt; Utilisation des outils open-source

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Mesurer RAN Peptide Toxicity à C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Utilisation de Caenorhabditis elegans pour dépister les interactions tissulaires spécifiques des chaperons

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...