Vorremmo riconoscere la natura collaborativa del laboratorio Hunter, e li ringrazio per la discussione è stata utile e l&#39;assistenza che ha reso lo sviluppo di questa tecnica possibile e divertente. Vorremmo ringraziare Nigel Delaney e il Caenorhabditis Genetica Centro per la vite senza fine e ceppi batterici. Questo lavoro è stato sostenuto dal National Institutes of Health GM089795 sovvenzione 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>

Nessun conflitto di interessi dichiarati.

Abbiamo qui presentato un nuovo metodo che permette l&#39;isolamento e la caratterizzazione di liquido extracellulare dal modello dell&#39;organismo C. elegans. La tecnica inizia con la manipolazione genetica o fisica di vermi donatori di aumentare il volume totale di fluido extracellulare. Liquido extracellulare è quindi isolato utilizzando una tecnica di microiniezione modificato. I vermi sono montati a secco per microiniezione utilizzando tamponi agarosio per tenere i vermi ancora durante la procedura. L&#39;attaccamento fisico al pad utilizza la potenza essiccare del pad asciutto per tenere idrostaticamente il worm al suo posto. Questo rappresenta una sfida, perché una volta che il worm è montato l&#39;acqua disponibile extracellulare comincia ad essere svuotato dagli animali donatori. E &#39;quindi indispensabile lavorare velocemente. Può essere utile per alcuni sperimentatori di sospendere i vermi in olio minerale sul pad iniezione, ma non li entrare in contatto con il pad. Dopo aver montato il pad iniezione su °microscopio elettronico, e allineando la vite senza fine e l&#39;ago attendere che il worm di entrare in contatto con il pad e l&#39;abbiano in &quot;monte&quot; in sé. Inoltre, anche se può essere possibile collocare fisicamente vermi su cuscinetti agar durante la microiniezione normale, troviamo che la forza fisica applicata per montare worm può notevolmente velocizzare l&#39;asciugatura di vermi donatori. E &#39;meglio prendere i vermi donatori in olio minerale, e trasferirli al pad mettendo la punta scelta vicino al pad e permettendo al worm di salire al largo del pick da solo. Oltre a lavorare rapidamente, è anche necessario per eseguire la tecnica vampirica isolamento pulito per evitare l&#39;infezione. L&#39;introduzione di batteri benigni tipicamente all&#39;interno della cuticola porta ad una infezione persistente e letale. Corretta tecnica elimina il verificarsi di infezione. Si può verificare la pulizia del trasferimento liquido extracellulare utilizzando batteri che possono essere facilmente identificati. Nello sviluppo della tecnica abbiamo trovato beneficial di praticare con ceppi batterici che possono essere seguiti da fluorescenza 4, 5. Se la contaminazione batterica entra il verme donatore viene facilmente osservata entro 24 ore dall&#39;infezione. Troviamo che i vermi destinatario mostrano un fenotipo più debole RNAi che i vermi donatori. Questo non è sorprendente come i vermi dei donatori sono coltivate su batteri che generano una fonte costante di dsRNA, mentre i destinatari di trasferimento di fluido pseudocoelomic sono sempre una singola dose a concentrazioni fisiologiche normali. È pertanto necessario considerare la limitazione di sensibilità e massimizzare la forza di RNAi nel donatore. L&#39;uso di IPTG per indurre la produzione dsRNA non si comporta in modo prevedibile con più induzione pari più efficiente RNAi. Segnaliamo ai lettori di caratterizzazione pubblicato di efficienza ambientale RNAi (cfr. 6,7,8). Suggeriamo che si determina sperimentalmente il metodo più efficiente della consegna al pr donatoreIOR di isolare liquido extracellulare per l&#39;analisi. Abbiamo dimostrato che i segnali sistemici silenziamento RNAi alimentato da dsRNA può essere rilevato nel liquido extracellulare. Caratterizzazione di questi segnali hanno dimostrato che l&#39;identificazione e l&#39;analisi genetica di liquido extracellulare è possibile. Anche se abbiamo utilizzato principalmente analisi genetica per caratterizzare questi segnali extracellulari, dovrebbe anche essere possibile effettuare analisi molecolare del fluido extracellulare. Fluido estratto può essere trattata, chimicamente o enzimaticamente, prima del trasferimento. Inoltre, il fluido da animali multiple possono essere raccolti in una micro-centrifuga provetta per analisi molecolare. Messa in comune di materiale da più donatori per il trasferimento in un singolo donatore è difficile, anche se può risultare possibile con la pratica. Con la raffinatezza e l&#39;adattamento si prevede future applicazioni che vanno ben al di là di ulteriore caratterizzazione di RNAi indurre segnali extracellulari. L&#39;identificazione e la caratterizzazione diRNA endogeni segnalazione sistemiche, analisi genetica dello sviluppo di vie di segnalazione intracellulari, l&#39;ambiente ei cambiamenti indotti nella composizione extracellulare può essere possibile adattando questa tecnica.

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Giorno</strong</td&gt;<td<strong&gt; Worm Prep</strong</td&gt;<td<strong&gt; Materiale Prep</strong</td&gt;</tr&gt;<tr&gt;<td&gt; -7 O prima</td&gt;<td&gt; Mantenere pulito, ben nutrito<em&gt; Clr-1 (e1745)</em&gt; E N2 vermi</td&gt;<td&gt; Accertarsi che gli elettrodi di iniezione, piatti, NGM NGM + Carb / piastre di IPTG, OP50 magazzino LB</td&gt;</tr&gt;<tr&gt;<td&gt; -6</td&gt;<td</td&gt;<tdStreak&gt; out RNAi cibo su LB + Carb</td&gt;</tr&gt;<tr&gt;<td&gt; -5</td&gt;<td</td&gt;<td&gt; Inoculare 3 mL durante la notte con RNAi cibo</td&gt;</tr&gt;<tr&gt;<td&gt; -4</td&gt;<td&gt; Sposta embrioni per RNAi piastra</td&gt;<td&gt; Seed RNAi piastra</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; Spostare vermi a 25 per 4 ore</td&gt;<td&gt; Crea piastre di saggio</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; Sposta worm per pulire le piastre NGM</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; Vampiric trasferimento</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td</td&gt;<td&gt; Recupera destinatario</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; +1</td&gt;<td&gt; Rimuovi destinatario del trasferimento</td&gt;<td</td&gt;</tr&gt;<tr&gt;<td&gt; +2</td&gt;<td&gt; Risultato Progeny</td&gt;<td</td&gt;</tr&gt;</tbody&gt;</table&gt;<p class="jove_content"<strong&gt; Tabella 1.</strong&gt; Timeline Preparazione del materiale.</p&gt;<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Attrezzatura</strong</td&gt;<td<strong&gt; Azienda</strong</td&gt;<td<strong&gt; Numero di catalogo</strong</td&gt;</tr&gt;<tr&gt;<td&gt; Picolitri pressione Iniettore</td&gt;<td&gt; Warner Instruments</td&gt;<td&gt; 65-0001 (PLI-100)</td&gt;</tr&gt;<tr&gt;<td&gt; Flaming / Brown micropipetta estrattore</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; Reagenti</strong&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; Olio minerale</td&gt;<td&gt; EM Scienza</td&gt;<td&gt; MX1560-1</td&gt;</tr&gt;<tr&gt;<td&gt; 22x50 n ° 1 ½ vetro di copertura</td&gt;<td&gt; Corning</td&gt;<td&gt;</td&gt;</tr&gt;<tr&gt;<td&gt; SeaKem LE agarosio</td&gt;<td&gt; Lonza</td&gt;<td&gt; 50004</td&gt;</tr&gt;<tr&gt;<td&gt; Vetro borosilicato Capillari</td&gt;<td&gt; Mondo Precisazioni Instruments</td&gt;<td&gt; 1B100F-4</td&gt;</tr&gt;<tr&gt;<td&gt; Soluzione di ipoclorito di sodio (5% di cloro attivo)</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; E ceppi batterici</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; Il Caenorhabditis Genetics Center (CGC)</td&gt;<td&gt; CB3241</td&gt;</tr&gt;<tr&gt;<td<em&gt; GLP-1</em&gt; RNAi vettore<sup&gt; 10</sup&gt;</td&gt;<td&gt; Fonte Bioscience (Biblioteca Alimentazione Ahringer)</td&gt;<td&gt; F02A9.6</td&gt;</tr&gt;<tr&gt;<td<em&gt; Pal-1</em&gt; RNAi vettore<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; Il 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; Tabella 2.</strong&gt; Reagenti e attrezzature specifiche.</p

vampiric isolation of extracellular fluid from caenorhabditis elegans

Il modello di organismo geneticamente trattabili C. elegans ha fornito dei chiarimenti in una miriade di questioni biologiche, attivata per il suo tempo di generazione breve, la facilità di crescita e di piccole dimensioni. Questa piccola dimensione, però, è non riconosciute una serie di approcci tecnici che si trovano in altri sistemi modello. Ad esempio, le trasfusioni di sangue nei sistemi di mammiferi e le tecniche di innesto delle piante consentono domande di composizione del sistema circolatorio e di segnalazione. Il sistema circolatorio del worm, il pseudoceloma, finora è stato impossibile per il dosaggio direttamente. Per rispondere alle domande di segnalazione intercellulare e circolatorio sistema di composizione C. elegans ricercatori hanno tradizionalmente rivolto a un&#39;analisi genetica, cellule / tessuti soccorso specifico e analisi mosaico. Queste tecniche forniscono un mezzo per inferire ciò che avviene tra le cellule, ma non sono universalmente applicabili identificazione e caratterizzazione di molecole extracellulari. Qui vi presentiamo una newly tecnica sviluppata per dosaggio direttamente il fluido pseudocoelomic di C. elegans. La tecnica inizia con uno manipolazione genetica o fisica per aumentare il volume di fluido extracellulare. Successivamente gli animali sono sottoposti ad una tecnica di microiniezione vampirica inversa usando un&#39;attrezzatura microiniezione che permette il controllo della pressione ammenda equilibrio. Dopo l&#39;isolamento del fluido extracellulare, il fluido raccolto può essere saggiata mediante trasferimento in altri animali o per mezzo molecolari. Per dimostrare l&#39;efficacia di questa tecnica presentiamo un approccio dettagliato per analizzare un esempio specifico di molecole segnale extracellulari, dsRNA lungo durante una risposta sistemica RNAi. Anche se la caratterizzazione di RNAi sistemica è una prova di principio esempio, vediamo come questa tecnica adattabile a rispondere a una serie di domande di composizione del sistema circolatorio e di segnalazione.

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

Il modello di organismo<em&gt; C. elegans</em&gt; Utilizza fluido pseudocoelomic come sistema circolatorio passiva. Saggio diretto di questo fluido non è stato possibile in precedenza. Qui presentiamo una nuova tecnica per direttamente dosaggio spazio extracellulare, e utilizzare segnali silenziamento sistemici durante una risposta RNAi come prova di principio esempio.

Isolamento del Vampiro di liquido extracellulare da<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

Research

JoVE Journal

Biology

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

10.1K Views.  Harvard University. Il modello di organismo C. elegans Utilizza fluido pseudocoelomic come sistema circolatorio passiva. Saggio diretto di questo fluido non è stato possibile in precedenza. Qui presentiamo una nuova tecnica per direttamente dosaggio spazio extracellulare, e utilizzare segnali silenziamento sistemici durante una risposta RNAi come prova di principio esempio.

Video: 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.

Misura Caenorhabditis elegans Life Span su terreni solidi

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

Ricerca

Biologia

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.

Isolamento e In vitro Attivazione di 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 ...

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.

Solida piastra a base di restrizioni alimentari, 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.

Microscopia time-lapse di Embriogenesi All&#39;inizio del 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.

Di base<em&gt; Caenorhabditis elegans</em&gt; Metodi: sincronizzazione e di osservazione

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.

Prostaglandina estrazione e l&#39;analisi 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, ...

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 supplementazione alimentare di acidi grassi polinsaturi a<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.

Misurazione dei tassi di consumo di ossigeno in intatto 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.

Purificazione e analisi di 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.

Quantificazione del declino proteostatico tessuto-specifico nella Caenorhabditis elegans

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