Wir möchten dankt Herrn Dr. Antonio Miranda-Vizuete für seine wertvolle Hilfe wollen. Wir möchten auch unseren Dank an Salma Boulayoune und Isabel Caballero Ausdruck für wertvolle technische Hilfe. Diese Arbeit wurde durch einen Zuschuss von der Junta de Andalucía (BIO-272) finanziert. Diese Forschung wurde in Absprache mit den aktuellen Gesetzen genetische Experimente in Europa durchgeführt.

1: Osmotische Vermeidung Assay. <ol><li> Über 16-24 Stunden vor dem Test, Pick L4 Larvenstadium Tiere jeder Genotyp eine frische NGM-Platte mit OP50 E. coli andincubate es bei 20 ° C. Am nächsten Tag starten das Experiment mit jungen Erwachsenen. </li><li> Es wird empfohlen, die Durchführung des Tests &quot;blind&quot;. Die Platten mit jeder Stamm zu untersuchenden sollte durch einen zweiten Experimentator neu etikettiert werden, der Durchführung des Tests mit dem neu etikettiert Platten, entlarvt als das Experiment beendet werden würde. </li><li> Der Tag des Tests, bereiten Sie einen 4M Stammlösung von Fructose mit 1% Kongorot Lösung auflösen und bei Raumtemperatur. Wir empfehlen, die Lösung vor jedem Test, da der Farbstoff mit der Zeit ausfallen könnte. </li><li> Kreisring (1 cm Durchmesser) auf NGM befindet sich auf der Mitte der festen Medium beschrieben, mit 15 ul der roten 4M Fructose-Lösung. Lassen Sie die Fructose-Lösung in den Agar einweichen, dies dauert in der Regel zwischen 2 und 5 Minuten. </li><li> Legen Sie einzelne junge erwachsene Tiere von jedem Stamm in den Ring und folgen in den nächsten 10 Minuten, um die Antwort auf die osmotische Barriere zu bestimmen. Tiere Vermeidung der Ring mehr als sechs Mal in Folge als normal eingestuft werden; Ausscheidenden am Ring in weniger als sechs Versuche sind als fehlerhaft in osmotische Empfindlichkeit. Hinweis: Die Steuerung Stammes muss in jedem Assay verwendet werden. N2 junge erwachsene Tiere werden als positive Kontrollen verwendet, da sie zu vermeiden entscheidend die Ringwallanlage. Tiere, die zuvor verhungert, ging durch Dauer-Larven oder aus Platten, die zu trocken kommen sollten nicht verwendet werden. Zu Beginn sollte der Kontrolltiere in zweifacher Ausfertigung ausgewertet werden, um zu bestätigen, dass die Assay-Platten und die Lösung korrekt sind. </li></ol> 2: Generierung von Knockdown Würmer durch RNAi Fütterung. <ol><li> RNAi Platten: NGM RNAi Fütterung Platten enthält pro Liter: 17 g Agar, 2,5 g Pepton, 3,0 g NaCl, 1 ml 5 mg ml -1 Cholesterin; füllen die Flasche zu 1 Liter mit H 2 O und Autoklaven. Nach Agar kühlt auf etwa 65 ° C, 25 ml 1 M KPO 4, pH 6,0, 1 ml 1 M CaCl 2, 1 ml 1M MgSO 4, 0,5 ml Carbenicillin (50 mg ml -1) und 1 ml 1M IPTG. Wenn Sie mit vorbereiteten soliden NGM beginnen, schmelzen festes Medium durch die Mikrowelle und anschließend mit Flüssigkeiten NGM auf der Bank zu kühlen und fügen Sie dann KPO 4, pH 6,0, CaCl 2, MgSO 4, Carbenicillin und IPTG wie zuvor angegeben. 10 ml NGM in jede 60 mm Petrischale. Abkühlen lassen und umkehren Platten Beibehaltung über Nacht bei RT vor dem Gebrauch. Die Platten können in einer Plastiktüte bei 4 ° C für ca. 4-5 Tage gelagert. </li><li> Bakterielle und Einleitung: Isolieren Sie Kolonien von E.coli HT115 (DE3)-Stamm, mit L4440 Vektor, der ein Fragment, das dem Ziel-Gen transformiert, auf Luria-Bertani (LB)-Agarplatten (17 g Agar, 10 g Trypton, 5 g Hefeextrakt und 10 g NaCl pro Liter) mit Ampicillin (50 ug / ml) und Tetracyclin (15 ug / ml). Wählen Sie eine Kolonie von Bakterien und impfen in LB mit 50 pg / ml Ampicillin, gewachsen und für 6 8 h unter Schütteln bei 37 ° C; Samen ein Tropfen dieser Kultur auf die vorbereiteten Platten NGM und trocknen die Platten gründlich, bevor sie Inkubation über Nacht ( 12 24 h) bei Raumtemperatur, damit die Bakterien wachsen und der Induktion zu beginnen. Die Inkubation wurde im Dunkeln, weil Tetracyclin ist lichtempfindlich und kann die Variabilität der RNAi-Assays unter Platten beeinflussen. Es ist notwendig, um eine positive und eine negative Kontrolle für RNAi Fütterungsversuche verwenden. Die positive Kontrolle ist E.coli HT115 Zelle mit der L4440 Vektor, der die unc-22-Gen-Sequenz umgewandelt. Der Knockdown von unc-22-Gen produziert ein &quot;Zucken Phänotyp&quot;. Die negative Kontrolle ist E.coli HT115 Zelle mit leeren L4440 transformiert. </li><li> Worm Handhabung und Scoring: Der erste Tag, Ort L4 Stadium Würmer aus einer NGM-Platte mit OP50 auf NGM-Platten ohne Bakterien und inkubieren ausgesät bei 20 ° C für 12 Stunden (Fasten). Am nächsten Tag, Transfer jungen Erwachsenen fasteten Zwitter auf einen Teller mit den Bakterien Ausdruck der spezifischen Zielgens RNAi seedded. Lassen Sie 40-48 Stunden bei 20 ° C bis F1-Nachkommen zu erzeugen. Dann legen Sie ein paar F1 erwachsenen Würmer auf eine andere Platte mit der gleichen Bakterien geimpft. Nach 24-48 Stunden, auszuwählen und zu isolieren, aus der F2-Nachkommen, junge Erwachsene (voll ausgebildete überstehende Vulva mit wenigen Eier) und Note für Phänotypen. Hinweis: der RNAi-Induktion in Neuronen haben einige Einschränkungen, weil der feuerfesten Eigenschaften der C elegans Nervensystem RNAi.. Zur Überwindung der Probleme dieser Ineffizienz in Neuron ist es empfehlenswert, die GebO-3-Stamm, eine hypersensible Hintergrund der Auswirkungen der RNAi in Neuronen 10 zu verwenden. In unseren Experimenten keine differences wurden zwischen Bristol N2 und RRF-3-Stämme für die Gene gefunden und den Phänotyp analysiert. </li></ol> 3: Die Stämme verwendet. Die C. elegans Stämme in dieser Arbeit sind in Tabelle 1 dargestellt. Mutanten-Stämme wurden gekreuzt gegen N2 Wildtyp - mindestens vier Mal, um unerwünschte zufällige Mutationen, die während der Mutagenese Protokoll erzeugt worden sein könnten, zu entfernen. .. OP50 E-coli-Stamm wurde von Caenorhabditis Genetic Center, University of Minnesota, USA E.coli HT115 (DE3) mit dem Plasmid pL4440 Durchführung NRX-1 (JA: C29A12.5) suppliied und NLG-1 (JA: C40C9.5 Gen)-Fragmente wurden durch Dr. Peter Askjaer, Centro Andaluz de Biología del Desarrollo (CABD), CSIC Universidad Pablo Olavide, Sevilla, Spanien zur Verfügung gestellt. 4: Repräsentative Ergebnisse. Illustrative Ergebnisse sind in den Abbildungen 1 und 2 dargestellt. Zehn Tiere von jedem Stamm und ein Minimum von drei Replika-Experimente wurden durchgeführt. <img alt="Abbildung 1" src="/files/ftp_upload/1616/1616Fig1.jpg"/> Abbildung 1. Experimente des osmotischen Vermeidungsverhalten. Control-Stämme und NRX-1 (ok1649 und tm1961) V mangelhaft mutantworms durch Umkehren rückwärts, wenn sie einen osmotischen Barriere (4M Fructose) Begegnung reagieren. NLG-1 (ok259 und tm474) X-Mutanten nicht, diese Barriere zu erkennen. Doppel-Mutanten in NLG-1 und NRX-1, Stämme CRR21 (ok1649; ok259) VX und CRR23 (tm1961; ok259) VX erholte sich der Wildtyp-Phänotyp. Der * zeigt signifikante Unterschiede (P ≤ 0,001) von t-Student-Test, in der Antwort von jedem Stamm der Bristol N2 Wildtyp von t-Student-Test <img alt="Abbildung 2" src="/files/ftp_upload/1616/1616Fig2.jpg"/> Abbildung 2. Experimente mit Knockdown Würmer durch RNAi Fütterung. E.coli HT115 (DE3) mit leeren pL4440 Vektor transformierte oder mit einem Fragment, das dem Zielgene NRX-1 oder NLG-1 wurden gefüttert anderer Wurm Stämme verwendet. Der * zeigt signifikante Unterschiede (P ≤ 0,001) von t-Student-Test, in der Reaktion des N2-Stamm mit Bakterien, die die pL4440 vector mit einem Fragment Targeting der NLG-1-Gens im Vergleich mit dem N2-Stamm mit leerem Vektor pL4440 eingespeist .

<ol>
	<li>Sudhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 455 (7215), 903-903 (2008).</li><li>Fabrichny, I. P., Leone, P., Sulzenbacher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 56 (6), 979-979 (2007).</li><li>Garber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Science. 317 (5835), 190-190 (2007).</li><li>Wang, K., Zhang, H., Ma, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 459 (7246), 528-528 (2009).</li><li>Ward, S., Thomson, N., White, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Journal of comparative neurology. 160 (3), 313-313 (1975).</li><li>Bargmann, C. I., Thomas, J. H., 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=Cold+Spring+Harbor+symposia+on+quantitative+biology.">Cold Spring Harbor symposia on quantitative biology.</a> 55, 529-529 (1990).</li><li>White, J. G., Southgate, E., Thomsom, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1-1 (1986).</li><li>Culotti, J. G., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Genetics. 90 (2), 243-243 (1978).</li><li>Fire, A., Xu, S., Montgomery, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 391 (6669), 806-806 (1998).</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=.">.</a> Nature. 395 (6705), 854-854 (1998).</li><li>Simmer, F., Tijsterman, M., Parrish, S., Koushika, S. P., Nonet, M. L., Fire, A., Ahringer, J., Plasterk, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Curr Biol. 12, 1317-1317 (2002).</li><li>Missler, M., Zhang, W., Rohlmann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. 423 (6943), 939-939 (2003).</li><li>Varoqueaux, F., Aramuni, G., Rawson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuron. 51 (6), 741-741 (2006).</li><li>Graf, E. R., Zhang, X., Jin, S. X., Scheiffele, P., Fan, J., Choih, 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> Cell. 119 (7), 1013-1013 (2004).</li><li>Scheiffele, P., Fan, J., Choih, 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> Cell. 101 (6), 657-657 (2000).</li><li>Jamain, S., Quach, H., Betancur, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 34 (1), 27-27 (2003).</li><li>Szatmari, P., Paterson, A. D., Zwaigenbaum , L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature genetics. 39 (3), 319-319 (2007).</li></ol>

Neurexine und Neuroligine übernehmen wichtige Rollen in der synaptischen Übertragung 11 und Differenzierung von synaptischen Verbindungen 12. Beide Moleküle haben als Kandidatengene für Autismus 13,14 identifiziert worden. In diesem Video zeigen wir eine einfache Methode, die uns um die Wirkung der Gene, die den osmotischen Vermeidung Antwort in C. Studie erlaubt elegans. Neuroligin Mutanten defekt sind bei der Aufdeckung von osmotischen...

Tabelle 1. C. elegans Stämme. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Belastung </td><td> Gen </td><td> Allele </td><td> Quelle </td></tr><tr><td> Bristol N2 </td><td> - </td><td> - </td><td> ÖCGK </td></tr><tr><td> VC228 </td><td> NLG-1 </td><td> ok259 </td><td> CGC </td></tr><tr><td> FX00474 </td><td> NLG-1 </td><td> tm474 </td><td> bNBP-JAPAN </td></tr><tr><td> VC1416 </td><td> NRX-1 </td><td> ok1649 </td><td> CGC </td></tr><tr><td> FX1961 </td><td> NRX-1 </td><td> tm1961 </td><td> NBP-JAPAN </td></tr><tr><td> NL2099 </td><td> GebO-3 </td><td> pk1436 </td><td> CGC </td></tr><tr><td> CRR21 </td><td> NRX-1; NLG-1 </td><td> ok1649; ok259 </td><td> Diese Arbeit </td></tr><tr><td> CRR22 </td><td> NRX-1; NLG-1 </td><td> tm1961; ok259 </td><td> Diese Arbeit </td></tr></tbody></table> a. Caenorhabditis Genetic Center, University of Minnesota, USA. b. Nationale Bioresource Projekt für das Experimental Animal &quot;Nematoden C. elegans&quot;. Tokyo Womens Medical University, Japan.

osmotic avoidance in caenorhabditis elegans: synaptic function of two genes, orthologues of human nrxn1 and nlgn1, as candidates for autism

Neurexine und Neuroligine sind Zelladhäsionsmoleküle in erregenden und hemmenden Synapsen, und sie sind für die korrekte neuronales Netzwerk-Funktion 1 erforderlich. Diese Proteine ​​sind an der präsynaptischen und postsynaptischen Membranen 2 gefunden. Studien an Mäusen zeigen, dass Neurexine und neurologins eine wesentliche Rolle in der synaptischen Übertragung 1 haben. Jüngste Berichte haben gezeigt, dass einer veränderten neuronalen Verbindungen während der Entwicklung des menschlichen Nervensystems könnten die Basis für die Ätiologie der zahlreichen Fälle von Autismus-Spektrum-Störungen 3 bilden. Caenorhabditis elegans konnte als experimentelles Werkzeug, um die Untersuchung der Funktionsweise der synaptischen Komponenten zu erleichtern verwendet werden, wegen seiner Einfachheit für Labor-Experimente, und da sein Nervensystem und synaptische Verkabelung wurde vollständig charakterisiert. In C. Elegans NRX-1 und NLG-1-Gene sind orthologen die menschliche NRXN1 und NLGN1 Gene, die alpha-Neurexin-1 und Neuroligin-1-Proteine, bzw. zu codieren. Bei Menschen und Nematoden, ist die Organisation von Neurexine und Neuroligine ähnlich in Bezug auf die funktionellen Domänen. Der Kopf des Nematoden enthält die amphid, ein Sinnesorgan des Nematoden, die Antworten vermittelt auf verschiedene Reize, einschließlich osmotische Stärke. Die amphid ist von 12 sensorischen Neuronen bipolar mit Flimmerepithel Dendriten und ein Axon präsynaptischen Terminal 4. Zwei von diesen Neuronen, genannt ASHR und ASHL sind besonders wichtig in osmotischen sensorische Funktion, Aufdeckung wasserlösliche Repellents mit hohen osmotischen Stärke 5. Die Dendriten der beiden Neuronen an der Spitze des Mundes zu verlängern und die Axone zu verlängern, um den Nerv Ring, wo sie synaptische Verbindungen mit anderen Neuronen Bestimmung der Verhaltensreaktion 6 machen. Zur Beurteilung der Auswirkungen der Neurexin und Neuroligin in hohen osmotischen Kräfte Vermeidung, zeigen wir die verschiedenen Reaktion von C. elegans-Mutanten in NRX-1 und NLG-1-Gene, mit einer Methode auf einem 4M Fructose Ring 7 basiert. Die Verhaltens-Phänotypen wurden bestätigt mit spezifischen RNAi-Klone 8. In C. elegans, kann die dsRNA erforderlich, um RNAi auslösen durch die Fütterung 9 verabreicht werden. Die Lieferung der dsRNA durch Nahrung induziert die RNAi Interferenz des Gens von Interesse so dass die Identifizierung von genetischen Komponenten und Netzwerk-Pfade.

Watch this Scientific Journal Video about Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism at JoVE.com

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

Neurexine und Neuroligine sind Membran-Neuron Haftung Proteine, die wichtige Rollen in synaptic Differenzierung und die Übertragung durchzuführen. Neuroligin defizienten Mutanten von<em&gt; C. elegans</em&gt; Defekt sind bei der Aufdeckung von osmotischen Kraft, aber wenn sie enthalten auch eine Mutation im Gen Neurexin, erholen sie den Wildtyp-Phänotyp.

Osmotische Vermeidung in Caenorhabditis elegans: Synaptic Function von zwei Genen, Orthologe der Menschenrechte NRXN1 Und NLGN1, Die als Kandidaten für Autismus

Neurexins and neuroligins are cell adhesion molecules present in excitatory and inhibitory synapses, and they are required for correct neuron network ...

osmotic-avoidance-caenorhabditis-elegans-synaptic-function-two-genes

Research

JoVE Journal

Biology

Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

11.7K Aufrufe.  Facultad de Ciencias, Universidad de Córdoba. Neurexine und Neuroligine sind Membran-Neuron Haftung Proteine, die wichtige Rollen in synaptic Differenzierung und die Übertragung durchzuführen. Neuroligin defizienten Mutanten von C. elegans Defekt sind bei der Aufdeckung von osmotischen Kraft, aber wenn sie enthalten auch eine Mutation im Gen Neurexin, erholen sie den Wildtyp-Phänotyp.

Serie: Osmotic Avoidance in Caenorhabditis elegans: Synaptic Function of Two Genes, Orthologues of Human NRXN1 and NLGN1, as Candidates for Autism

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.

Isolation und In-vitro- Aktivierung von Caenorhabditis elegans Sperma

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

Forschung

Biologie

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.

Zeitraffer-Mikroskopie der frühen Embryogenese 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 ...

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.

Grundlegende<em&gt; Caenorhabditis elegans</em&gt; Methoden: Synchronisation und 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 ...

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

Biochemische und High Throughput Mikroskopische Beurteilung der Fettmasse in<em&gt; Caenorhabditis elegans</em

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

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.

Prostaglandin Extraktion und Analyse in<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, ...

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 ...

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.

Nahrungsergänzung von mehrfach ungesättigten Fettsäuren<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, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

der Anbau von<em&gt; Caenorhabditis elegans</em&gt; In drei Dimensionen im Labor

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Probenvorbereitung und Bildgebung von Exosomen von Transmissions-Elektronenmikroskopie

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

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.

Messung von Sauerstoff Verbrauchsmengen in intakten Caenorhabditis elegans

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