Name: dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans
Uploaded: 2013-11-29T16:00:00
Description: Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Vi takker Chris Webster til at udføre de indledende forsøg er vist i figur 3 i de repræsentative resultater, og Jason Watts og Chris Webster for nyttige kommentarer til manuskriptet. Finansieringen af ​​denne undersøgelse blev leveret af en bevilling fra National Institutes of Health (USA) (R01DK074114) til JLW. Nogle nematode stammer, der anvendes i dette arbejde blev leveret af Caenorhabditis Genetik Center, som er finansieret af NIH Office of Research Infrastructure Programmer (P40 OD010440).

Flerumættede fedtsyrer er følsomme over for varme, lys og oxygen. Derfor skal der udvises forsigtighed ved udarbejdelsen af ​​fedtsyrer tilskud plader, således at fedtsyrer, ikke udsættes for overskydende varme og lys. NGM medier indeholdende 0,1% Tergitol (NP-40) autoklaveres og afkøles delvist, hvorefter fedtsyre natriumsalte tilsættes under konstant omrøring. Pladerne får lov til at tørre i mørke. Optagelse af fedtsyrer ved C. elegans dyrket på disse plader kan derefter blive overvåget ved ga...

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Her beskriver vi en fremgangsmåde til supplering af C. elegans med kosten umættede fedtsyrer. Som nævnt ovenfor, skal man passe på i udarbejdelsen af PUFA suppleret plader fordi den reaktive karakter af dobbeltbindinger i flerumættede fedtsyrer forårsager disse fedtsyrer for at være følsomme over for oxidation gennem varme og lys 11. For at undgå oxidation, er det vigtigt at tilsætte PUFA&#39;en til væsken agarmedium, når mediet er afkølet til 55 ° C og lagrer plader i et mørkt miljø....

Fedtsyrer er essentielle strukturelle komponenter af membraner samt en effektiv energilagerindretning molekyler. Derudover kan fedtsyrer spaltes fra cellemembranerne af lipaser og enzymatisk modificeret til at producere signalerer effektorer 1. Naturligt forekommende polyumættede fedtsyrer (PUFA) indeholder to eller flere cis-dobbeltbindinger. Omega-3-fedtsyrer og omega-6 fedtsyrer er skelnes fra hinanden baseret på positionerne af dobbeltbindinger med hensyn til methylenden af ​​fedtsyren. Sund kost kræver både omega-6 og omega-3 fedtsyrer. Men vestlige kost er særligt rige på omega-6 fedtsyrer og fattig på omega-3 fedtsyrer. En høj omega-6 omega-3 fedtsyre-forhold er forbundet med øget risiko for hjerte-kar-og inflammatoriske sygdomme, er imidlertid de præcise gavnlige og skadelige funktioner af specifikke fedtsyrer ikke godt forstået 2. Spolorm Caenorhabditis Elegans er nyttig i at studere fedtsyre funktion, fordi det syntetiserer alle de enzymer, der er nødvendige for at producere en række af omega-6 og omega-3-fedtsyrer, herunder en omega-3 desaturase, en aktivitet, der er fraværende i de fleste dyr 3,4. Mutanter, der mangler fedtsyredesaturase-enzymer ikke producere specifikke PUFA&#39;er, hvilket fører til en række udviklingsmæssige og neurologiske defekter 4-6. At studere de fysiologiske effekter af kosten fedtsyrer, har vi udviklet et biokemisk assay kompatibel med genetisk analyse ved hjælp af både mutant og RNAi knock-down teknikker i C. elegans. Tilskud af specifikke PUFA&#39;er er opnået ved tilsætning af en fedtsyre natriumsaltopløsning til agarmediet før udhældning. Dette resulterer i PUFA optagelse af E. coli fødekilde, når den akkumuleres i bakterielle membraner. C. elegans indtager de PUFA-holdige bakterier, og dette kosttilskud er tilstrækkelig til at redde defekttværsnit af PUFA-mutanter. Tilskud af de fleste fedtsyrer har ingen skadelige virkninger på vildtype dyr dog specifikke omega-6 fedtsyrer, især dihomo-gamma linolensyre (DGLA, 20:03 n-6) forårsage en permanent ødelæggelse af C. elegans kønsceller 7,8. Gaschromatografi bruges til at overvåge udbredelsen af ​​det supplerede fedtsyre i bakterielle fødekilde (enten OP50 eller HT115) samt i nematoder. Tilsætningen af detergent Tergitol (NP-40) i medierne giver mulighed for jævn fordeling af fedtsyrer gennem hele pladen og mere effektiv optagelse af fedtsyrer ved E. coli og nematoder. Vi har fundet, at umættede fedtsyrer let optages af bakterier og C. elegans, men optagelsen af mættede fedtsyrer er meget mindre effektiv. Denne artikel vil beskrive trin-for-trin, hvordan man supplere agar medier med fedtsyrer, samt hvordan man kan overvåge fedtsyre optagelse i the nematode hjælp gaskromatografi.

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	<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

Fedtsyrer er essentielle for mange cellulære funktioner. De tjener som effektive energilagring molekyler, gøre op den hydrofobe kerne af membraner, og deltage i forskellige signalveje. Caenorhabditis elegans syntetiserer alle de enzymer, der er nødvendige for at producere en række af omega-6 og omega-3 fedtsyrer. Dette, kombineret med det enkle anatomi og vifte af tilgængelige genetiske redskaber, gør det til en attraktiv model for at studere fedtsyre funktion. For at undersøge den genetiske veje, der medierer de fysiologiske virkninger af kosten fedtsyrer, har vi udviklet en metode til at supplere C. elegans kost med umættede fedtsyrer. Tilskud er et effektivt middel til at ændre fedtsyresammensætningen af ​​orme og kan også anvendes til at redde defekter i fedtsyrer-deficient mutanter. Vores metode anvender nematodevækst medium agar (NGM) suppleret med fedtsyre acidsodium salte. Fedtsyrerne i suppleret pladerne bliver integreringented i membraner i bakterielle fødekilde, som derefter taget op af C. elegans, der lever af de suppleret bakterier. Vi beskriver også en gaskromatografi protokol til at overvåge ændringer i fedtsyresammensætningen som opstår i suppleret orme. Dette er en effektiv måde at supplere kosten til både store og små bestande af C. elegans, der giver mulighed for en række applikationer til denne metode.

Supplering af C. elegans diæt er begrænset af evnen af den bakterielle fødekilde til optagelse og indarbejde fedtsyre i det bakterielle membran. At bestemme evnen af E. coli OP50 at assimilere forskellige fedtsyrer i sine membraner, OP50 blev udpladet på medier uden tillæg, 0,1 mM og 0,3 mM koncentrationer af stearinsyre (18:00), natriumoleat (18:01 n-9), og natrium-DGLA (20 : 3n-6). Pladerne blev tørret ved stuetemperatur i 2 dage i mørke, og inkuberet ved 20 ° C i 3 dage. Bakterielle græspl?...

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 elegan &lt;/ em&gt; er en nyttig model til at udforske funktionerne i flerumættede fedtsyrer i udvikling og fysiologi. Denne protokol beskriver en effektiv metode til at supplere &lt;em&gt; C. elegans &lt;/ em&gt; kost med flerumættede fedtsyrer.

Kosttilskud af polyumættede fedtsyrer i<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, ...

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Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

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 ...

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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 ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Grundlæggende<em&gt; Caenorhabditis elegans</em&gt; Metoder: Synkronisering og Observation

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Prostaglandin Udvinding og Analyse i<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

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

dyrkning af<em&gt; Caenorhabditis elegans</em&gt; I tre dimensioner i laboratoriet

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 ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Måling af ilt forbrug satser i intakt 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

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

Rensning og analyse af Caenorhabditis elegans Ekstracellulære 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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Kvantificering af vævsspecifik proteostatisk tilbagegang i caenorhabditis eleganer

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

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Modellering Alders-associerede neurodegenerative sygdomme i Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...