Nous remercions sciences de la vie et de la Faculté des arts et des sciences de l&#39;Université Queen pour le financement de ces travaux. De plus, nous remercions le laboratoire Chin-Sang pour fournir les réactifs nécessaires, l&#39;équipement et le soutien technique. Nous remercions également QGEM 2011 surtout Tony Il pour sa contribution à la discussion.

1. Préparation / laver les Worms <ol><li> Synchroniser vers de jeune adulte 11. </li><li> Pipette 2 ml de la Basal S sur une plaque de Chemotaxis de 5 cm de scène vers qui viennent défrichées la pelouse du OP50 E. coli. Inclinaison de la plaque en fonction des besoins pour assurer les vers sont lavées à partir de la surface de la plaque dans la mémoire tampon. </li><li> Pipette 1 ml de la solution Basal ver-S dans un tube de centrifugation. </li><li> Centrifuger pendant 10 secondes à l&#39;aide d&#39;un PicoFuge à 6600 rpm. </li><li> Aspirer le Basal S, laissant le culot de vers non perturbées. </li><li> Ajouter 1 ml solution Basal S au tube de centrifugation, la retournant plusieurs fois de se laver les vers. </li><li> Répétez les étapes 1.4 à 1.6 encore trois fois. </li><li> Centrifuger pendant 10 secondes une cinquième fois, cette fois aspirer le surnageant dans un volume final de ca. 100 pi. </li><li> Pipette 2 pl des vers sur une plaque NGM pour s&#39;assurer qu&#39;il n&#39;y a entre 50 et 250 vers dans chaque échantillon 2 pl. Réglerla concentration de vers dans le Basal S au besoin en remettant en suspension les vers dans un volume plus petit ou plus grand de S basale. </li><li> Utilisation des vers dans l&#39;essai immédiatement après le lavage des vers pendant jusqu&#39;à 1 heure. </li></ol> 2. Préparation des plaques d&#39;essai <ol><li> Marquant la face inférieure d&#39;une plaque 5 cm, il faut diviser le plat en 4 quadrants égaux. agar Chemotaxis ou NGM peuvent être utilisés. Un minimum de 3 essais sont requis pour chaque souche génétique testé. </li><li> Marquez un cercle de rayon de 0,5 cm autour de l&#39;origine (Figure 1). </li><li> Marquer un point dans chaque quadrant soit avec un &quot;T&quot; pour &quot;Test&quot; ou &quot;C&quot; pour &quot;configuration&quot;, assurant que les sites sont à égale distance de l&#39;origine et de l&#39;autre. Les points doivent être d&#39;au moins 2 cm de l&#39;origine. Marquer le coin supérieur gauche et inférieur droit quadrants comme quadrants de test et en haut à droite et en quadrants en bas à gauche que les contrôles (Figure 1). </li></ol> 3. Exécution de l&#39;essai </p&gt; <ol><li> Mélanger la solution d&#39;essai en combinant des volumes égaux de composé d&#39;essai et de 0,5 M d&#39;azoture (anesthésique utilisé pour arrêter les vers de terre après avoir atteint un quart de cercle). Mélanger la solution de commande en combinant le solvant utilisé pour diluer le composé d&#39;essai avec 0,5 M azoture. </li><li> Pipette 2 pl de la solution de ver (préparé en 1.8) de la pastille sur l&#39;origine (où les deux lignes se croisent). </li><li> Immédiatement après, pipette 2 pl de la solution de test sur les deux sites &quot;t&quot;. De même, une pipette la même quantité de la solution de contrôle sur les deux sites «C». </li><li> Une fois le ver et odorant gouttes ont été absorbés dans l&#39;agar, remplacez les couvercles et retournez les plaques. </li><li> Après 60 minutes, placez les vers dans un 4 ° C incubateur. Ne retirez une plaque de l&#39;incubateur quand il peut être compté. Laissant les vers à la température ambiante peut leur permettre de mobiliser à nouveau. </li><li> Notez le nombre de vers dans chaque quadrant qui a complètement traversé la circulaire intérieurele. </li><li> Répéter les étapes 3.2 à 3.6 à l&#39;aide de la solution de commande dans les deux quadrants du test et de contrôle. Cela servira de la plaque de commande. Trois de ces plaques doivent être prises et exécutées pour servir un contrôle approprié. </li></ol> 4. Interprétation des Scores / Détermination de l&#39;indice Chemotaxis <ol><li> Calculer l&#39;indice de chimiotactisme en utilisant l&#39;équation 1. Cela donnera un indice chimiotactique entre -1.0 et +1.0. </li></ol> (1) Index Chemotaxis = (# Worms inscrites dans les quadrants d&#39;essai - # Worms dans les deux quadrants de contrôle) / (Nombre total de Worms marqués) Un score +1.0 indique attraction maximale vers la cible et représente 100% des vers qui arrivent dans les quadrants contenant la cible chimique. Un index de -1,0 est la preuve de répulsion maximale. Méthodes d&#39;analyse similaires ont déjà été employées 3 (Tableau 1). <ol start="2"><li> Signaler le Chemotaxi moyennes Index (CI) et l&#39;erreur standard de la moyenne (SEM). Effectuez le test t de Student comparant les données des plaques d&#39;essai et de contrôle. </li></ol>

<ol>
	<li>Bargmann, C. I., Hartwieg, 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=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74, 515-527 (1993).</li><li>Bargmann, C. I., 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=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7, 729-742 (1991).</li><li>Lee, J., Jee, C., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+preference+in+C.+elegans.">Ethanol preference in C. elegans.</a> Genes Brain Behav. 8, 578-585 (2009).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat. Methods. 8, 592-598 (2011).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 817-821 (1973).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14, 803-812 (1995).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Troemel, E. R., Kimmel, B. E., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+chemotaxis+responses:+sensory+neurons+define+olfactory+preferences+in+C.+elegans.">Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans.</a> Cell. 91, 161-169 (1997).</li><li>Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+pirouettes+in+Caenorhabditis+elegans+chemotaxis.">The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.</a> J Neurosci. 19, 9557-9569 (1999).</li><li>Frokjaer-Jensen, C., Ailion, M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium-acetate+is+sensed+by+gustatory+and+olfactory+neurons+in+Caenorhabditis+elegans.">Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans.</a> PLoS One. 3, e2467 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+Methods:+Synchronization+and+Observation.">Basic Caenorhabditis elegans Methods: Synchronization and Observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Sengupta, P., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+specification+and+differentiation+in+the+nervous+system+of+Caenorhabditis+elegans.">Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans.</a> Dev. Genet. 18, 73-80 (1996).</li><li>Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> WormBook. , (1895).</li></ol>

Nous n&#39;avons rien à révéler.

Chemotaxis, bien que contrôlé par un ensemble complexe de mécanismes neuronaux et cellulaire, peut être facilement et objectivement quantifiée en utilisant des dosages de chimiotaxie. Pour obtenir les meilleurs résultats des tests, certaines étapes cruciales doivent être prises. Tout d&#39;abord, la mise en scène des vers est essentiel à donner des résultats expérimentaux cohérents. Worms à différents stades de la vie comportent différemment 13; vers la scène de manière mixte peut fausser le...

Ward abord développé le test de chimiotactisme en 1973 5, et depuis lors il a eu des applications de grande envergure. Neurobiologie est un domaine qui a bénéficié de l&#39;aide d&#39;une variété d&#39;essais de chimiotaxie. Adaptation olfactive, une forme simple d&#39;apprentissage et la mémoire, a été démontrée dans C. elegans utilisant des dosages de chimiotaxie 6. Ils ont également été utilisés pour montrer que C. elegans peuvent développer l&#39;éthanol tolérance un résultat qui démontre non seulement la plasticité comportementale des vers, mais qui montre aussi que les vers peuvent être très utiles dans l&#39;étude de la dépendance à l&#39;alcool chez l&#39;homme 3. Les analyses ont même été élaborés pour démontrer la capacité de C. elegans de stocker court et la mémoire à long terme, en montrant que les associations sont faites par les vers entre chemoattractants et alimentaires (OP50) 7. En outre, étant donné les nombreuses informations disponibles au sujet de la C. elegans génome, le chemotaxile comportement de C. elegans a été modifié à plusieurs reprises par l&#39;induction de mutations 1,8. Cela permet de nombreuses possibilités d&#39;ingénierie passionnants, tels que le développement de C. elegans comme un outil de bioremédiation. Ainsi, depuis le développement initial de l&#39;essai de chimiotaxie en 1973, il a été souvent modifié et utilisé pour élucider des mystères dans une variété de disciplines. Certains tests visant à découvrir l&#39;itinéraire spécifique prise par les vers vers une cible. Le test prototype de ce genre a été développé par Ward 5. Trois vers ont été placés sur gélose fondue pendant 15 min. Leurs mouvements ont été tracées par l&#39;empreinte qu&#39;ils ont quitté alors qu&#39;ils se rendaient à la périphérie de la plaque jusqu&#39;à un dégradé à un appât au centre de la plaque. Tous les vers sur la plaque ont été arrêtés avec du chloroforme à la fin de chaque essai. Un descendant de cette méthode d&#39;une seule vis sans fin placée dans le milieu de la plaque avec l&#39;attractif et le contrôle d&#39;unt distances égales et opposées de l&#39;origine 2. Pierce-Shimomura et al. développé un test pour observer la nature exacte du mouvement impliqué dans le chimiotactisme 9. Vers individuelles ont été placés dans des boîtes de Pétri de 9 cm soit contenant une concentration uniforme de l&#39;appât ou un dégradé radial en forme, aboutissant à la source de l&#39;attractif. Un programme de logiciel d&#39;ordinateur qui a reconnu la vis sans fin est utilisé pour enregistrer le comportement observé. Une caméra vidéo fixée à un microscope travaille en conjonction avec l&#39;étape d&#39;ajuster automatiquement la boîte de Pétri que l&#39;essai de court à assurer le ver est resté dans le champ de vision. Sur cette base, des informations plus détaillées a été découvert sur ​​la cause de pirouettes affichées par C. elegans. D&#39;autres épreuves, plus semblable à celle décrite ici, tester la réponse d&#39;une population de vers de composés à tester. Dosages de chimiotaxie deux quadrants ont étéutilisés pour explorer les rôles que les différents neurones, des récepteurs et des molécules de transduction des signaux lus lorsque C. elegans ont été exposés à divers composés 1. Entre 20 à 50 vers la chaux ont été placés à proximité du centre de la plaque avec une substance attractive et d&#39;un contrôle au niveau des extrémités polaires ainsi que l&#39;anesthésique, l&#39;azoture de sodium (NaN3). Après 60 min, un indice chimiotactique avec des valeurs de -1,0 à 1,0 a été générée sur la base de la différence entre le nombre de vers de terre ont été fixés sur l&#39;attractif ou la commande. Un indice chimiotactique similaire a été utilisée dans le dosage indiqué dans cet article, bien que le test plus tôt n&#39;a pas réussi à évaluer rigoureusement les vers non mobiles. Ce test a ensuite été appliqué à la suite de tester les effets de l&#39;ablation neuronale sur la chimiotaxie. Une autre variante de l&#39;essai ci-dessus a été réalisée de 100 à 200 où les vers ont été placés au centre d&#39;une plaque contenant quatre quadrants 3. Quadrants adjacents contenaient soit le test ousubstance contrôler. Comme dans les tests précédents, les vers ont été immobilisées par l&#39;action de l&#39;azoture de sodium, avant d&#39;être marqué. Une méthode similaire est décrite ici comme un moyen d&#39;évaluer la réponse de C. elegans à différents composés. Cependant, la méthode ci-dessous présente l&#39;avantage supplémentaire de seulement évaluer les vers qui ont passé un seuil de distance séparant mobile à partir vers immobiles. D&#39;autres essais ont incorporé des directives similaires pour ignorer les vers immobiles. Frøkjær-Jensen et al. mis au point un test polyvalent qui peut être utilisé pour tester des composés solubles à la fois volatiles et l&#39;eau 10. Une boîte de Pétri a été divisé en quatre quadrants. Les quadrants du haut et du bas ne contiennent pas de solvants. Le quadrant gauche contenait de l&#39;eau, et le droit contenait la substance attractive. Lors du test de substances odorantes volatiles, l&#39;analyte a été placé sur le couvercle de la boîte, sur le quadrant approprié, tandis que les composés solubles dans l&#39;eau ont été placés directement sur l&#39;agar. Les méthodes actuellement en vigueur pour l&#39;évaluation de la réponse chimiotactique de C. elegans sont constamment affinés pour optimiser leur facilité d&#39;utilisation, l&#39;efficacité et la précision. Ainsi, alors que le test décrit ici a la capacité d&#39;évaluer le plus grand nombre de vers (débit maximal:. 250 vers / heure par plaque, légèrement supérieur au débit démontré par Lee et al 3); la véritable force de cette méthode est l&#39; succinct aboutissement de plusieurs des attributs d&#39;essais antérieurs (tableau 1).

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH2O to 1 liter.] Autoclave</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PicoFuge</td>
 <td>Stratagene</td>
 <td>400552</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscopes</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P1000 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P10 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M Sodium Azide</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chemotaxis Agar [1.6% BBL-agar (Benton-Dickinson) or 2% Difco-agar. Autoclave. Add 5 mM potassium phosphate, pH 6.0; 1 mM CaCl2, 1 mM MgSO4]</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethanol/Distilled Water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Test Compounds (eg. 0.5% diacetyl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Bio-Rad</td>
 <td>166-0600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>Amresco</td>
 <td>CA97062-046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>VWR</td>
 <td>CA12001-120</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4OH</td>
 <td>BDH</td>
 <td>CABDH8641-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.25% Tween 20</td>
 <td>Bio-Rad</td>
 <td>170-6531</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

c. elegans chemotaxis assay

De nombreux organismes utiliser chemotaxis à rechercher des sources de nourriture, éviter les substances nocives, et trouver des partenaires. Caenorhabditis elegans a un comportement de chimiotactisme impressionnant. La prémisse de tester la réponse des vers à une substance odorante est de les placer dans une zone et observer le mouvement évoquée en réponse à une substance odorante. Même avec les nombreux tests disponibles, l&#39;optimisation ver lieu de départ par rapport à la fois le contrôle et d&#39;essai, tout en minimisant l&#39;interaction du virus avec l&#39;autre, tout en conservant une taille importante de l&#39;échantillon reste un travail en cours 1-10. La méthode décrite ici a pour but de remédier à ces problèmes en modifiant le dosage développée par Bargmann et al. 1. Une boîte de Pétri est divisé en quatre quadrants, deux quadrants opposés marqués &quot;Test&quot; et deux sont désignés &quot;Control&quot;. Anesthésique est placé dans tous les essais et les sites témoins. Les vers sont placés au centre de la plaque avec une Circle marquée autour de l&#39;origine de veiller à ce que les vers non mobiles seront ignorés. Utilisant un système à quatre quadrants plutôt que 2 ou deux 1 élimine biais dans le mouvement des vers, car ils sont à égale distance à partir d&#39;échantillons de test et de contrôle, quel que soit le côté de l&#39;origine, ils ont commencé. Cela évite le problème de vers peuvent être contraints de voyager à travers un ensemble d&#39;autres vers de terre pour répondre à une substance odorante, ce qui peut retarder les vers ou les forcer à prendre une voie plus détournée, ce qui donne une interprétation erronée de leur trajectoire prévue. Cette méthode présente également des avantages pratiques en ayant une taille d&#39;échantillon plus large et permettant au chercheur d&#39;exécuter le test sans surveillance et marquer les vers une fois le temps imparti est écoulé.

Comparaison de type sauvage (N2) C. elegans à l&#39;ODR-10 (KY10) mutant. Nous avons utilisé le diacétyle, un C. connu elegans chimiotactique et de comparer les vers de type sauvage à celui d&#39;un mutant qui manque le récepteur de diacétyle 1,12. Par type sauvage (N2) vers l&#39;index chimiotactique était 0,100 ± 0,066 pour l&#39;éthanol et 0,839 ± 0,031 à 0,5% diacétyle. Comme prévu, le diacétyle provoque une réponse chim...

Watch this Scientific Journal Video about C. elegans Chemotaxis Assay at JoVE.com

C. elegans Chemotaxis Assay

Procédé d&#39;évaluation quantitative de la réponse chimiotactique des<em&gt; Caenorhabditis elegans</em&gt; Est décrite. Un indice chimiotactique (CI) a été utilisé comme un moyen d&#39;évaluer précisément la réponse des vers de terre pour certaines cibles, et de servir de plate-forme de comparaison entre les souches et les composés d&#39;intérêt.

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Research

JoVE Journal

Biology

45.0K vues.  Queen's University. Procédé d'évaluation quantitative de la réponse chimiotactique des Caenorhabditis elegans Est décrite. Un indice chimiotactique (CI) a été utilisé comme un moyen d'évaluer précisément la réponse des vers de terre pour certaines cibles, et de servir de plate-forme de comparaison entre les souches et les composés d'intérêt.

Vidéo: C. elegans Chemotaxis Assay

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Génération de Stable elegans transgéniques C. Utilisation de microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Recherche

Biologie

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 ...

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

Paradigmes pour la caractérisation pharmacologique de C. elegans mutants transmission synaptique

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Dissection et d&#39;enregistrement de la jonction neuromusculaire C. Elegans

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Génération de souris transgéniques C. elegans Par transformation biolistique

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

Dépistage ARNi pour identifier les phénotypes postembryonnaires dans C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

Un procédé de culture embryonnaire<em&gt; C. elegans</em&gt; Cellules

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

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 ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Mesurer les effets des bactéries sur<em&gt; C. Elegans</em&gt; Comportement aide d&#39;un test de rétention d&#39;oeufs

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...