Ringraziamo Chris Webster per l&#39;esecuzione degli esperimenti preliminari mostrati in Figura 3 dei risultati rappresentativi e Jason Watts e Chris Webster per gli utili commenti sul manoscritto. Finanziamento per questo studio è stato fornito da una sovvenzione da parte del National Institutes of Health (USA) (R01DK074114) per JLW. Alcuni ceppi di nematodi utilizzati in questo lavoro sono stati forniti dal Caenorhabditis Genetics Center, che è finanziato dall&#39;Ufficio NIH di programmi di infrastrutture di ricerca (P40 OD010440).

Gli acidi grassi polinsaturi sono sensibili al calore, luce e ossigeno. Pertanto, occorre prestare attenzione quando si prepara piatti di integratori di acidi grassi tale che gli acidi grassi non siano esposti a eccesso di calore e di luce. Supporti NGM contenenti 0,1% Tergitol (NP-40) è autoclavato e parzialmente raffreddati, dopo che i sali sodici degli acidi grassi sono aggiunti con agitazione costante. Le piastre vengono lasciati asciugare al buio. Assorbimento di acidi grassi da C. elegans coltivati ​​su queste piastre possono poi essere monitorata mediante gascromatografia. 1. Preparazione di acidi grassi integrato multimediale <ol><li> Misurare componenti multimediali in un pallone di dimensioni adeguate. Per 1 L, aggiungere 17 g Bacto-agar, 2,5 g di triptone, 3 g di NaCl, 1 colesterolo ml 5 mg / ml disciolto in etanolo e 10 ml 10% tergitol disciolto in acqua. </li><li> Aggiungere 80% del volume finale di acqua desiderata Millipore e autoclave media con bottiglie di vetro vuoto (uguale al numero di differenticoncentrazioni di acidi grassi da testare), e anche opportuno cilindri graduati. </li><li> Media freddi in un bagnomaria regolato a 55 ° C. Mentre i media si raffredda preparare la soluzione madre di lavoro del sale di acido grasso di sodio rompendo aperta la fiala di vetro, utilizzando misure di sicurezza e garantire particelle di vetro non si mescolano con gli acidi grassi. Pesare acido grasso abbastanza per fare una scorta di lavoro 100 mm. </li><li> Portare la soluzione di acido grasso ad una concentrazione finale di 100 mM con acqua purificata. Sciogliere completamente il sale di sodio dell&#39;acido grasso richiede in genere circa 20-30 min. Spurgare il lavoro stock di acido grasso con argon o azoto, perché il contatto con un gas inerte impedisce l&#39;ossidazione. Tappare il flacone e conservare lo stock lavorare al buio fino a quando i media si è raffreddato. </li><li> (Facoltativo) Se si desidera RNAi supporto, misurare ampicillina e soluzioni isopropil β-D-1-thiogalactopyranoside (IPTG) qui. </li><li> Quando l&#39;agar è raffreddato a 55 ° C per aggiungere 1 L: 1ml di 1 M MgSO4, 1 ml di 1 M CaCl 2, e 25 ml di tampone fosfato (108,3 g KH 2 PO 4 e 35,6 g K 2 HPO 4 per 1 L autoclavato). Aggiungere la soluzione IPTG se la fabbricazione di piastre di RNAi filtro ampicillina sterilizzato e. Per tutti i tipi di piastre, aggiungere acqua sterile per portare il volume finale a supporti 1 L. </li><li> Vicino a una fiamma, trasferimento multimediale in un autoclave e di dimensioni adeguate cilindro graduato e aggiungere acqua sterile calda per volume desiderato. Media Transfer torna a pallone iniziale e mescolare agitando. </li><li> Vicino a una fiamma, trasferire file multimediali in un certo numero di bottiglie pari al numero di concentrazioni da testare misurando con una autoclavato e di dimensioni adeguate cilindro graduato. Mantenere i media aliquotate come liquido con un bagno di acqua fino a quando il lavoro stock acido grasso si è completamente dissolto. </li><li> Mettere una bottiglia su un piatto mescolare e mescolare fino a quando la media è calda al tatto, ma non calda. Assicurarsi di lasciare se stessi abbastanzatempo di suscitare nella soluzione stock di acidi grassi e versare le piastre prima della media comincia a solidificare. Se piastra di agitazione ha il controllo della temperatura, impostarlo a 55 ° C. </li><li> Diluire il lavoro stock acido grasso 100 mm nella media per la concentrazione finale desiderata. Supporti Mescolare finché il precipitato bianco è in soluzione (circa 1 minuto). Il materiale può essere ancora leggermente torbida dopo. </li><li> (Facoltativo) Se RNAi supporto si desidera aggiungere l&#39;ampicillina a 0,1 mg / ml e IPTG a 2 mm di concentrazione finale. </li><li> Versare memoria utilizzando un aiuto pipetta automatizzata e una sterile 25 ml pipetta, aggiungere 8 ml di media per 60 millimetri piastra o 25 ml di mezzi per mm piastra 100. Ripetere aggiunta di acido grasso e piastra versando passaggi per i restanti bottiglie. </li><li> Dopo l&#39;agar è solidificato, coprire le piastre acidi grassi completata con una scatola o conservare a temperatura ambiente in un cassetto ben ventilati per proteggere dall&#39;ossidazione luce. </li><li> Seed E. coli OP50 sulle piastre, due giorni dopo piastre sono secchi.Per piastre 60 mm, pipetta 300 microlitri di una cultura OP50 overnight (coltivato a 37 ° C). Se sono stati versati piatti RNAi, seminare con 300 ml di batteri RNAi dopo che le piastre si sono asciugati per quattro giorni. Piatto tempo di asciugatura può essere necessario regolare a causa di differenze di umidità in vari ambienti di laboratorio. </li><li> Incubare le piastre in un ambiente buio a temperatura ambiente, mentre il prato batterica sta asciugando. </li></ol> 2. Indurre cellule germinali distruzione da supplementazione di DGLA <ol><li> C. elegans coltivate su piastre contenenti 0,3 DGLA mm (20:03 n-6) diventano sterili a causa della distruzione delle cellule germinali in entrambi palco e adulti nematodi larvali. <ol><li> Preparare una popolazione sincronizzato di L1 larve trattando ermafroditi gravide con soluzione di ipoclorito alcalino (per una soluzione di 10 ml: 0,5 ml di NaOH 5 M, 2,5 ml di candeggina, e 7 ml H 2 O). Agitare delicatamente gli ermafroditi gravide in questa soluzione fino a quando il verme adultos dissolvenza. Le uova saranno conservati e possono essere pellet per centrifugazione a bassa velocità. </li><li> Lavare preparazione uovo 3 volte in tampone M9 (3 g di KH 2 PO 4, 6 g di Na 2 HPO 4, 5 g NaCl, 1 ml di 1 M MgSO 4, H 2 O a 1 L. Aggiungi MgSO 4 dopo la sterilizzazione in autoclave). Per fornire abbastanza ossigeno, risospendere le uova in un tubo di plastica da 15 ml per un volume finale di 5 ml di tampone M9 e roccia su un agitatore notte. </li><li> L1 larve deve essere aggiunto alle piastre, completata due giorni dopo la semina con OP50, o un giorno dopo placcatura batteri RNAi. Incubare vermi a 20 ° C per tre giorni fino a raggiungere lo stadio adulto. </li></ol></li><li> Vermi adulti possono essere lanciati per la sterilità utilizzando un microscopio da dissezione con abbastanza alto ingrandimento per visualizzare gli embrioni in via di sviluppo in utero. Perdita di cellule germinali successo apparirà come un utero chiaro vuoto di uova. </li><li> I worm possono anche essere ottenuto mediante la fissazione e colorazione con la nucleico uncid colorante diamindinophenylindole (DAPI), che facilita la visualizzazione dei nuclei nelle cellule germinali e embrioni di sviluppo. Una macchia DAPI rapida si ottiene prelevando vermi in una goccia di tampone M9 su un vetro d&#39;orologio 9. <ol><li> Flood vetro di orologio con 1 ml di 0,2 ng / m. DAPI in soluzione di etanolo al 95%, lasciar riposare per circa 5 min. </li><li> Scegli vermi in una goccia di Vectashield su un vetrino, e poi coprire con un vetrino. </li><li> Seal vetrino e conservare a 4 ° C al buio durante la notte per la colorazione ottimale, tuttavia diapositive possono anche essere visualizzati immediatamente. Punteggio di presenza o assenza di nuclei di cellule germinali usando un microscopio a fluorescenza equipaggiato con una lampada UV e filtro. </li></ol></li></ol> 3. Confermando Fatty Acid assorbimento da gascromatografia Nel complesso composizione di acidi grassi C. elegans possono essere determinate con la produzione di esteri metilici degli acidi grassi (FAME), che vengono poi separati e quantificate usando chr gasomatography 4. <ol><li> Raccogliere 500-1000 vermi adulti da loro lavando via delle piastre con acqua e trasferimento in un silanizzata 13 millimetri x 100 millimetri tubo tappo a vite in vetro. </li><li> Lasciate che i vermi si depositano per gravità, e quindi rimuovere quanta più acqua possibile con una pipetta Pasteur di vetro. </li><li> Lavare worm una volta con acqua, e poi di nuovo eliminare quanta più acqua possibile. </li><li> FAMEs sono formati aggiungendo 1 ml di 2,5% H 2 SO 4 in metanolo, quindi riscaldamento a 70 ° C per 1 ora in un bagno d&#39;acqua. </li><li> Rimuovere i tubi dal bagnomaria e lasciare raffreddare per 1 min. </li><li> Estrarre FAMEs aggiungendo 1,5 ml di acqua e 0,25 ml di esano. </li><li> Tubi Recap e agitare vigorosamente. </li><li> Provette da centrifuga in una centrifuga da tavolo clinico per 1 min alla massima velocità per separare esano dal solvente acquoso. </li><li> Trasferire esano (strato superiore) per un inserto GC fiala in una fiala GC, facendo attenzione a non trasferire qualsiasi della fase acquosa. Per GC unanalisi, 1-2 ml di FAMEs in esano viene iniettato in un capillare gascromatografia in colonna polare adatto per l&#39;analisi FAMEs. Il Agilent 7890 GC iniettore è fissata a 250 ° C, con una portata di 1,4 ml / min, e il forno GC è programmato per una temperatura iniziale di 130 ° C, che è tenuto per 1 min. Successivamente, la temperatura viene rampa 10 ° C / min fino a 190 ° C, e poi rampa di nuovo a 5 ° C / min fino a 210 ° C e mantenuta ancora per 1 min. </li><li> Per garantire è verificato che l&#39;assorbimento di DGLA, analizzare FAMEs dalla ionizzazione di fiamma rilevamento (FID) o spettrometria di massa (MS) rilevazione, utilizzando standard autentici per l&#39;identificazione del C. elegans acidi grassi. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Qui si descrive un metodo di supplementazione di C. elegans con acidi grassi insaturi nella dieta. Come accennato in precedenza, è necessario prestare attenzione nella preparazione di piatti PUFA, completata perché la natura reattiva dei doppi legami in PUFA causa questi acidi grassi siano sensibili all&#39;ossidazione mediante calore e luce 11. Per evitare l&#39;ossidazione, è importante aggiungere il PUFA al mezzo liquido agar dopo che il supporto è raffreddato a 55 ° C e memorizza le piastre ...

Acidi grassi sono componenti strutturali essenziali delle membrane nonché molecole di immagazzinamento dell&#39;energia efficiente. Inoltre, gli acidi grassi possono essere scissi dalla membrane cellulari da lipasi e essere enzimaticamente modificati per produrre effettori di segnalazione 1. Naturalmente presenti acidi grassi polinsaturi (PUFA) contengono due o più doppi legami cis. Gli acidi grassi omega-3 e gli acidi grassi omega-6 si distinguono tra loro in base alle posizioni di legami doppi rispetto alla fine metilico dell&#39;acido grasso. Le diete sane richiedono sia gli acidi grassi omega-3, omega-6 e. Tuttavia, le diete occidentali sono particolarmente ricchi di omega-6 acidi grassi e povera di acidi grassi omega-3. Un alto omega-6 rapporto omega-3 acidi grassi è associata ad aumentato rischio di malattie cardiovascolari ed infiammatorie, tuttavia, le precise funzioni benefici e dannosi degli acidi grassi specifici non sono ben compresi 2. Gli ascaridi Caenorhabditis Elegans è utile nello studio funzione acido grasso perché sintetizza tutti gli enzimi necessari per produrre una gamma di acidi omega-6 e omega-3 grassi, compreso un omega-3 desaturasi, un&#39;attività che è assente nella maggior parte degli animali 3,4. Mutanti privi di grassi acido enzimi desaturasi non riescono a produrre PUFA specifici, portando ad una serie di difetti di sviluppo e neurologici 4-6. Per studiare gli effetti fisiologici degli acidi grassi alimentari, abbiamo sviluppato un saggio biochimico compatibile con l&#39;analisi genetica utilizzando sia tecniche di RNAi di knock-down mutante e in C. elegans. Supplementazione con PUFA specifici è ottenuto aggiungendo una soluzione di sale di sodio di acido grasso per mezzo di agar prima di versare. Ciò si traduce in PUFA assorbimento della E. fonte di cibo coli, dove si accumula nelle membrane batteriche. C. elegans ingerire i batteri PUFA contenenti, e questa integrazione alimentare è sufficiente per salvare il difettosots di mutanti PUFA-deficienti. La supplementazione della maggior parte degli acidi grassi ha effetti negativi sul tipo di animali selvatici, tuttavia, specifici acidi grassi omega-6, in particolare dihomo-acido gamma linolenico (DGLA, 20:03 n-6), causare una distruzione permanente di C. elegans cellule germinali 7,8. Cromatografia gas è utilizzato per monitorare l&#39;assorbimento dell&#39;acido grasso integrato nella fonte alimentare batterica (sia OP50 o HT115) nonché nei nematodi. L&#39;aggiunta del tergitol detersivo (NP-40) nei mezzi permette una distribuzione uniforme degli acidi grassi attraverso l&#39;intera piastra e l&#39;assorbimento più efficiente degli acidi grassi della E. coli e nematodi. Abbiamo trovato che gli acidi grassi insaturi sono prontamente assorbiti da batteri e C. elegans, ma l&#39;assorbimento di acidi grassi saturi è molto meno efficiente. Questo articolo descrive passo-passo come integrare il supporto agar con acidi grassi, e come monitorare l&#39;assorbimento degli acidi grassi in the nematodi mediante gascromatografia.

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

Gli acidi grassi sono essenziali per numerose funzioni cellulari. Servono molecole di accumulo di energia come efficienza, costituiscono il nucleo idrofobico delle membrane, e partecipare a varie vie di segnalazione. Caenorhabditis elegans sintetizza tutti gli enzimi necessari a produrre una gamma di acidi omega-6 e omega-3 acidi grassi. Questo, combinato con la semplice anatomia e varietà di strumenti genetici disponibili, lo rendono un modello attraente per studiare la funzione di acidi grassi. Per studiare i percorsi genetici che mediano gli effetti fisiologici degli acidi grassi alimentari, abbiamo sviluppato un metodo per integrare il C. elegans dieta con acidi grassi insaturi. L&#39;integrazione è un mezzo efficace per alterare la composizione in acidi grassi di vermi e può essere utilizzato anche per salvare difetti di mutanti di acidi carenti di grassi. Il nostro metodo utilizza nematode crescita agar (NGM) integrato con sali acidsodium grassi. Gli acidi grassi nelle piastre integrate diventano incorporazioneTED nelle membrane della fonte di cibo batterica, che viene poi ripreso dalla C. elegans che si nutrono di batteri integrati. Noi descriviamo anche un protocollo gas cromatografia per monitorare i cambiamenti nella composizione degli acidi grassi che si verificano in vermi completata. Questo è un modo efficace per integrare le diete di piccole e grandi popolazioni di C. elegans, consentendo una vasta gamma di applicazioni di questo metodo.

Integrazione del C. elegans dieta è limitato dalla capacità della fonte alimentare batterica all&#39;assorbimento e incorporare acidi grassi nella membrana batterica. Per determinare la capacità di E. coli OP50 assimilare diversi acidi grassi nelle sue membrane, OP50 è stato placcato su supporto senza supplemento, 0,1 mM e 0,3 mM concentrazioni di acido stearico (18:00), oleato di sodio (18:01 n-9), e sodio DGLA (20 : 3n-6). Le piastre sono state essiccate a temperatura ambiente per 2 giorni al bui...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

&lt;em&gt; Caenorhabditis elegant &lt;/ em&gt; è un modello utile per esplorare le funzioni di acidi grassi polinsaturi nello sviluppo e nella fisiologia. Questo protocollo descrive un metodo efficiente di integrare &lt;em&gt; C. elegans &lt;/ em&gt; dieta con acidi grassi polinsaturi.

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, ...

dietary-supplementation-polyunsaturated-fatty-acids-caenorhabditis

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

17.0K Views.  Washington State University.  Caenorhabditis elegant è un modello utile per esplorare le funzioni di acidi grassi polinsaturi nello sviluppo e nella fisiologia. Questo protocollo descrive un metodo efficiente di integrare C. elegans dieta con acidi grassi polinsaturi. 

Video: Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

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 ...

Ricerca

Biologia

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, ...

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.

La coltivazione di<em&gt; Caenorhabditis elegans</em&gt; In tre dimensioni in laboratorio

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

Combinato di nucleotidi e proteina estrazioni in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

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 ...

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Purificazione e analisi di Caenorhabditis elegans Extracellular Vesicles

Quantificazione del declino proteostatico tessuto-specifico nella Caenorhabditis elegans