Nous tenons à souligner la nature collaborative du laboratoire de Hunter, et les remercier pour le débat utile et d&#39;assistance qui fait du développement de cette technique possible et amusant. Nous tenons à remercier Nigel Delaney et le Caenorhabditis Genetics Center for ver et souches bactériennes. Ce travail a été soutenu par les Instituts nationaux de la santé GM089795 subvention à CPH.

<ol>
	<li>Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Stable+Transgenic+C.+elegans+Using+Microinjection.">Generation of Stable Transgenic C. elegans Using Microinjection.</a> J. Vis. Exp. (18), e833-e833 (2008).</li><li>Hirose, T., Nakano, Y., Nagamatsu, Y., Misumi, T., Ohta, H., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+GMP-dependent+protein+kinase+EGL-4+controls+body+size+and+lifespan+in+C+elegans.">Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans.</a> Development. 130, 1089-1099 (2003).</li><li>Kimble, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+cell+lineage+following+laser+ablation+of+cells+in+the+somatic+gonad+of+Caenorhabditis+elegans.">Alterations in cell lineage following laser ablation of cells in the somatic gonad of Caenorhabditis elegans.</a> Dev. Biol. 87, 286-300 (1981).</li><li>Hegreness, M., Shoresh, N., Hartl, D., Kishony, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+equivalence+principle+for+the+incorporation+of+favorable+mutations+in+asexual+populations.">An equivalence principle for the incorporation of favorable mutations in asexual populations.</a> Science. 311, 1615-1617 (2006).</li><li>Labrousse, A., Chauvet, S., Couillault, C., Kurz, C. L., Ewbank, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+is+a+model+host+for+Salmonella+typhimurium.">Caenorhabditis elegans is a model host for Salmonella typhimurium.</a> Curr. Biol. 10, 1543-1545 (2000).</li><li>Kamath, R. S., Martinez-Campos, M., Zipperlen, P., Fraser, A. G., 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=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome. Biol. 2, (2001).</li><li>Min, K., Kang, J., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+feeding+RNAi+method+for+simultaneous+knock-down+of+more+than+one+gene+in+Caenorhabditis+elegans.">A modified feeding RNAi method for simultaneous knock-down of more than one gene in Caenorhabditis elegans.</a> Biotechniques. 48, 229-232 (2010).</li><li>Zhuang, J. J., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specificity+of+Caenorhabditis+elegans+Enhanced+RNAi+Mutants.">Tissue-specificity of Caenorhabditis elegans Enhanced RNAi Mutants.</a> Genetics. 188 (1), 235-237 (2011).</li><li>Way, J. C., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mec-3,+a+homeobox-containing+gene+that+specifies+differentiation+of+the+touch+receptor+neurons+in+C.+elegans.">mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans.</a> Cell. 54, 5-16 (1988).</li><li>Kamath, R. S., Fraser, A. G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Bot, N. L. e., Moreno, S., Sohrmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+functional+analysis+of+the+Caenorhabditis+elegans+genome+using+RNAi.">Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.</a> Nature. 421, 231-237 (2003).</li><li>Winston, W. M., Molodowitch, C., Hunter, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+RNAi+in+C.+elegans+requires+the+putative+transmembrane+protein+SID-1.">Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1.</a> Science. 295, 2456-2459 (2002).</li></ol>

Aucun conflit d&#39;intérêt déclaré.

Nous avons présenté ici une nouvelle méthode qui permet l&#39;isolement et la caractérisation des liquides extracellulaires de l&#39;organisme modèle C. elegans. La technique commence par la manipulation génétique ou physique de vers donateurs à accroître leur volume total du liquide extracellulaire. Fluide extracellulaire est ensuite isolé en utilisant une technique de micro-injection modifiée. Les vers sont montés pour la micro-injection à l&#39;aide d&#39;agarose tampons secs pour emprisonner les vers encore au cours de la procédure. L&#39;attachement physique à la plage utilise le pouvoir desséchant du tampon sec à transmission hydrostatique tenir le ver en place. Cela représente un défi, car une fois que le ver est monté l&#39;eau disponible extracellulaire commence à être évacuée des animaux donneurs. Il est donc impératif de travailler rapidement. Il peut être bénéfique pour certains expérimentateurs de suspendre les vers dans l&#39;huile minérale sur le pavé d&#39;injection, mais ne pas avoir à prendre contact avec le tampon. Après le montage du coussinet d&#39;injection sur emicroscope électronique, et l&#39;alignement de la vis sans fin et une aiguille attendre le ver de prendre contact avec le patin et le faire «monter» lui-même. En outre, bien qu&#39;il puisse être possible de placer physiquement les vers sur des patins de gélose au cours de la micro-injection normale, nous constatons que la force physique appliquée à monter vers peuvent grandement accélérer le séchage des vers donateurs. Il est préférable de ramasser les vers des bailleurs de fonds dans l&#39;huile minérale, et de les transférer à la plage en mettant la pointe choix près du pavé et en permettant au ver de grimper hors de la camionnette sur son propre. En plus de travailler rapidement, il est également nécessaire de procéder à la technique d&#39;isolement vampirique proprement pour éviter l&#39;infection. L&#39;introduction de bactéries bénignes généralement à l&#39;intérieur de la cuticule conduit à une infection persistante et meurtrière. Une bonne technique élimine l&#39;apparition de l&#39;infection. On peut tester pour la propreté de transfert de fluide extracellulaire en utilisant des bactéries qui sont facilement identifiés. Lors du développement de la technique nous avons trouvé qu&#39;il bénéficiel de pratiquer avec des souches bactériennes qui peuvent être suivies par fluorescence 4, 5. Si la contamination bactérienne entre le ver des donateurs, il est facile d&#39;observer un délai de 24 heures après l&#39;infection. Nous constatons que les vers bénéficiaires montrent un phénotype plus faible que les ARNi vers des bailleurs de fonds. Cela n&#39;est pas surprenant que les vers des bailleurs de fonds sont cultivés sur des bactéries générant une source constante d&#39;ARN double brin, alors que les bénéficiaires de transfert de fluide pseudocoelomic reçoivent une dose unique à des concentrations physiologiques normales. Il est donc nécessaire d&#39;examiner la limitation de la sensibilité et de maximiser la force de l&#39;ARNi chez le donneur. L&#39;utilisation d&#39;IPTG pour induire la production ARNdb ne se comporte pas d&#39;une manière prévisible avec plus égale à induction plus efficace ARNi. Nous rappelons aux lecteurs de la caractérisation de l&#39;environnement publié ARNi efficacité (voir 6,7,8). Nous suggérons que l&#39;on détermine expérimentalement la méthode la plus efficace de livraison à la pr donateursIOR à isoler le liquide extracellulaire pour l&#39;analyse. Nous avons montré que les signaux systémiques silencieux ARNi de nourri ARNdb peuvent être détectés dans le liquide extracellulaire. La caractérisation de ces signaux démontré que l&#39;identification et l&#39;analyse génétique du liquide extracellulaire est possible. Bien que nous ayons principalement utilisé l&#39;analyse génétique pour caractériser ces signaux extracellulaires, il devrait également être possible d&#39;effectuer une analyse moléculaire du liquide extracellulaire. Fluide retiré peut être traité, par voie chimique ou par voie enzymatique, avant le transfert. En outre, le fluide de plusieurs animaux peuvent être recueillies dans un tube de centrifugeuse pour micro-dosage moléculaire. La mise en commun de matériel de donateurs multiples pour le transfert dans un seul donneur est difficile, mais peut-être possible avec la pratique. Avec perfectionnement et d&#39;adaptation que nous prévoyons de futures applications qui vont bien au-delà de caractérisation plus poussée de l&#39;ARNi induire des signaux extracellulaires. L&#39;identification et la caractérisation desendogènes ARN de signalisation systémiques, l&#39;analyse génétique des voies de signalisation intracellulaires de développement et les changements dans la composition induites par l&#39;environnement extracellulaire peut être possible en adaptant cette technique.

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Journée</strong</td&gt;<td<strongPréparation Worm&gt;</strong</td&gt;<td<strongPréparation Matériel&gt;</strong</td&gt;</tr&gt;<tr&gt;<td&gt; -7 Avant ou</td&gt;<td&gt; Maintenir propres, bien nourris<em&gt; Clr-1 (e1745)</em&gt; N2 et les vers</td&gt;<td&gt; Assurez-tampons d&#39;injection, plaques NGM, NGM + Carb / plaques IPTG, OP50 actions LB</td&gt;</tr&gt;<tr&gt;<td&gt; -6</td&gt;<td</td&gt;<tdStreak&gt; ARNi sur la nourriture sur LB + Carb</td&gt;</tr&gt;<tr&gt;<td&gt; -5</td&gt;<td</td&gt;<td&gt; Inoculer 3 mL nuit avec ARNi alimentaire</td&gt;</tr&gt;<tr&gt;<td&gt; -4</td&gt;<td&gt; Déplacez embryons à l&#39;ARNi plaque</td&gt;<td&gt; Graines ARNi plaque</td&gt;</tr&gt;<tr&gt;<td&gt; -3</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; -2</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; -1</td&gt;<td</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; 0</td&gt;<td&gt; Déplacer vers à 25 pendant 4 heures</td&gt;<td&gt; Faire des plaques d&#39;essai</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; Déplacer vers pour nettoyer les plaques NGM</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; Vampirique transfert</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; Récupérer destinataire</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; +1</td&gt;<td&gt; Retirer destinataire du transfert</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; +2</td&gt;<tdProgeny Score&gt;</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt; Tableau 1.</strong&gt; Calendrier Préparation du matériel.</p&gt;<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Matériel</strong</td&gt;<td<strong&gt; Société</strong</td&gt;<td<strongNuméro de catalogue&gt;</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Pression picolitres injecteur</td&gt;<td&gt; Warner Instruments</td&gt;<td&gt; 65-0001 (PLI-100)</td&gt;</tr&gt;<tr&gt;<td&gt; Flaming / Brown micropipette extracteur</td&gt;<td&gt; Sutter Instruments</td&gt;<td&gt; P97</td&gt;</tr&gt;<tr&gt;<td&gt; Axiovert 200</td&gt;<td&gt; Zeiss</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td colspan="3"<strong&gt; Réactifs</strong&gt;</td&gt;</tr&gt;<tr&gt;<tdHuile minérale&gt;</td&gt;<td&gt; EM Science</td&gt;<td&gt; MX1560-1</td&gt;</tr&gt;<tr&gt;<td&gt; 22x50 n ° 1 ½ verre Couvercle</td&gt;<td&gt; Corning</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; SeaKem LE Agarose</td&gt;<td&gt; Lonza</td&gt;<td&gt; 50004</td&gt;</tr&gt;<tr&gt;<td&gt; Borosilicate Glass capillaires</td&gt;<td&gt; Précisions du monde Instruments</td&gt;<td&gt; 1B100F-4</td&gt;</tr&gt;<tr&gt;<td&gt; Solution d&#39;hypochlorite de sodium (5% de chlore disponible)</td&gt;<td&gt; JT Baker</td&gt;<td&gt; 9416-01</td&gt;</tr&gt;<tr&gt;<td colspan="3"<strong<em&gt; C. elegans</em&gt; Souches bactériennes et</strong&gt;</td&gt;</tr&gt;<tr&gt;<td<em&gt; Clr-1 (e1745) II</em&gt;<sup&gt; 9</sup</td&gt;<td&gt; Le Caenorhabditis Genetics Center (CGC)</td&gt;<td&gt; CB3241</td&gt;</tr&gt;<tr&gt;<td<em&gt; Glp-1</em&gt; ARNi vecteur<sup&gt; 10</sup&gt;</td&gt;<td&gt; Source Bioscience (Bibliothèque alimentation Ahringer)</td&gt;<td&gt; F02A9.6</td&gt;</tr&gt;<tr&gt;<td<em&gt; Pal-1</em&gt; ARNi vecteur<sup&gt; 11</sup&gt;</td&gt;<td&gt;</td&gt;<td&gt; PHC187</td&gt;</tr&gt;<tr&gt;<td&gt; OP50-GFP<sup&gt; 5</sup&gt;</td&gt;<td&gt; Le Caenorhabditis Genetics Center (CGC)</td&gt;<td&gt; OP50-GFP</td&gt;</tr&gt;<tr&gt;<td&gt; YFP<em&gt; E. coli</em&gt;<sup&gt; 4</sup&gt;</td&gt;<td&gt;</td&gt;<td&gt; MC4100-YFP</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt; Tableau 2.</strong&gt; Réactifs et équipements spécifiques.</p

vampiric isolation of extracellular fluid from caenorhabditis elegans

Le maïs génétiquement docile organisme modèle C. elegans a fourni des indications sur une multitude de questions biologiques, a permis par son temps de génération court, la facilité de croissance et de petite taille. Cette petite taille, cependant, a refusé un certain nombre d&#39;approches techniques trouvées dans des systèmes modèles d&#39;autres. Par exemple, les transfusions sanguines dans les systèmes mammaliens et des techniques de greffage dans les plantes permettent de poser des questions de composition du système circulatoire et de signalisation. Le système circulatoire du ver, le pseudocoelom, a été jusqu&#39;à récemment impossible de doser directement. Pour répondre aux questions de signalisation intercellulaire et le système circulatoire composition C. chercheurs elegans ont traditionnellement recours à l&#39;analyse génétique, cellulaire / tissulaire spécifique de sauvetage, et l&#39;analyse de la mosaïque. Ces techniques fournissent un moyen pour déduire ce qui se passe entre les cellules, mais ne sont pas universellement applicable dans l&#39;identification et la caractérisation des molécules extracellulaires. Nous présentons ici un newly technique mis au point pour doser le fluide directement pseudocoelomic de C. elegans. La technique commence soit par manipulation génétique ou physique pour augmenter le volume de fluide extracellulaire. Ensuite, les animaux sont soumis à une technique de micro-injection vampirique inverse en utilisant une plate-forme de micro-injection qui permet bien de commande de pression d&#39;équilibre. Après isolement du liquide extracellulaire, le liquide recueilli peut être analysée par transfert dans d&#39;autres animaux ou par des moyens moléculaires. Pour démontrer l&#39;efficacité de cette technique, nous présentons une approche détaillée pour doser un exemple spécifique de molécules de signalisation extracellulaire, à long ARN double brin lors d&#39;une réponse systémique ARNi. Bien que la caractérisation de RNAi systémique est une preuve de principe par exemple, nous voyons cette technique comme étant adaptable pour répondre à une variété de questions de la composition du système circulatoire et la signalisation.

Watch this Scientific Journal Video about Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans at JoVE.com

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

L&#39;organisme modèle<em&gt; C. elegans</em&gt; Utilise un fluide pseudocoelomic comme un système circulatoire passive. Dosage direct de ce fluide n&#39;a pas été possible auparavant. Ici, nous présentons une nouvelle technique pour analyse directement l&#39;espace extracellulaire, et utiliser les signaux silencieux systémiques lors d&#39;une réponse ARNi comme une preuve de principe par exemple.

Isolation vampirique de fluide extracellulaire d&#39;<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

vampiric-isolation-of-extracellular-fluid-from-caenorhabditis-elegans

Nanyang Technological University

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Biology

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

10.1K vues.  Harvard University. L'organisme modèle C. elegans Utilise un fluide pseudocoelomic comme un système circulatoire passive. Dosage direct de ce fluide n'a pas été possible auparavant. Ici, nous présentons une nouvelle technique pour analyse directement l'espace extracellulaire, et utiliser les signaux silencieux systémiques lors d'une réponse ARNi comme une preuve de principe par exemple.

Vidéo: Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Mesure Caenorhabditis elegans</emDurée de vie> sur des milieux solides

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

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Biologie

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

L&#39;isolement et la In vitro Activation des Caenorhabditis elegans Sperme

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Solide plaque à base de restriction alimentaire Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse de microscopie du début de l&#39;embryogenèse dans les Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

De base<em&gt; Caenorhabditis elegans</em&gt; Méthodes: Synchronisation et d&#39;observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Extraction de prostaglandine et de l&#39;analyse en<em&gt; Caenorhabditis elegans</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

La supplémentation alimentaire en acides gras polyinsaturés dans<em&gt; Caenorhabditis elegans</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Mesure des taux de consommation d’oxygène intactes de Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

Purification et analyse de Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

Quantification du déclin protéostatique spécifique aux tissus chez Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...