Name: culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment
Uploaded: 2014-08-02T15:00:00
Description: Watch this Scientific Journal Video about Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment at JoVE.com

Dette arbejde blev støttet af National Institutes of HealthGrants DK85035 og DK074797 (IH).

1.. Worm Stammer <ol><li> Anskaf C. elegans vildtype Bristol N2 stammer fra Caenorhabditis Genetik Center (CGC) (http://www.cbs.umn.edu/cgc), og vedligeholde dem på NGM plader podet med E. coli-stamme OP50 7. Bemærk: Transgene orm stammer IQ6011 (Phrg-1 :: GFP) udnyttet blev genereret som tidligere beskrevet 11. IQ6011 kan rekvireres fra den tilsvarende forfatter. </li></ol> 2. Fremstilling af Modified C. elegans</e...

<ol>
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I denne protokol præsenterer vi en modificeret aksenisk flydende medier mCeHR der giver mulighed for hurtig C. elegans generation med produktion af et stort antal orme. Dette medie viser flere fordele som orme dyrkes uden at forurene E. coli eller bakterielle biprodukter og det kan udnyttes i ernæringsmæssige og toksikologiske undersøgelser. Anvendelsen af E. coli eller andre bakterier i sådanne undersøgelser har flere ulemper. For eksempel kan væksten af ​​bakterierne ændre under ...

Jordbunden rundorm, Caenorhabditis elegans, er en kraftig model organisme, der anvendes i en lang række undersøgelser fra genetik til toksikologi. Som et resultat af dens 1 mm størrelse, hurtige generationstid på fire dage, let dyrkning, og store afkom numre, har disse nematoder blevet udnyttet i en række genetiske og farmakologiske skærme 1, 2. Forskere drage fordel af denne orm til at identificere molekyler og veje bevaret i hvirveldyr-systemer. Disse veje omfatter celledød signaler, veje for aldring og stofskifte, og nervesystemet 3-6. Derudover gennemsigtighed C. elegans giver mulighed for generering af transgene linjer ved hjælp af fluorescerende protein reportere, som kan visualiseres direkte at analysere genekspression mønstre og protein lokalisering. I mange undersøgelser denne nematode dyrkes på en solid agarbaseret overflade ved hjælp nematode medium vækst (NGM) plader eller i liquid kulturer podes med Escherichia coli som en fødekilde 7,8. Disse bakterielle fødekilder kan forvirre biokemiske og toksikologiske undersøgelser med interferens fra bakteriel biprodukter påvirker fortolkningen af ​​resultaterne. For at undgå disse kompoundering virkninger C. elegans kan dyrkes i et axeniske flydende medier, der er blottet for bakterier som en fødekilde. Ved hjælp af dette medie, vi med succes dyrket millioner af meget synkroniserede orme for mange standard C. elegans protokoller herunder microarray analyse af differentielt regulerede gener i C. elegans udsat for forskellige hæm-koncentrationer, og produktionen af transgene orme anvender gen bombardement. Dette medie er kemisk definerede, og modificeret fra en original opskrift formuleret af Dr. Eric Clegg 9. Ved hjælp af denne mCeHR medier, har vi med succes identificeret gener involveret i hæm homeostase, kaldet hæm responsive gener (HRG er) 10, hvilket villehar ikke været muligt i regelmæssige vækstbetingelser, der anvender NGM agarplader podet med E. coli. I denne protokol beskriver vi proceduren for oprettelse og vedligeholde C. elegans dyrket på E. coli til aksenisk mCeHR og udnytte denne metode til at opnå et stort antal orme til fremstilling af transgene C. elegans linjer ved hjælp af mikropartikelbombardement. Præsenterer vi også undersøgelser, der viser nytten af at bruge axeniske medier til bestemmelse af ernæringsmæssige krav C. elegans hjælp hæm som et eksempel. Disse undersøgelser viser, at ved hjælp af mCeHR medier giver hurtig vækst af et stort antal af C. elegans for mange downstream-applikationer udnyttet af ormen forskere.

<table border="0" cellpadding="0" cellspacing="0" width="754">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">MgCl2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">M-2393</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">S-4641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium citrate.H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-1722</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CuCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C455-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MnCl2.4H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">M87-100</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ZnCl2</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">Z-0152</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Fe(NH4)2(SO4)2.6H2O</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-1018</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">CaCl2.2H2O</td>
			<td style="width:108px;">Fisher</td>
			<td style="width:121px;">C70-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5 -monophosphate, sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-1752</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytidine 5 -phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-1006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanosine 2 - and3&#160; -monophosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-8002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uridine 5 -phosphate, disodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">U-6375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymine&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T0376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">N-Acetylglucosamine</td>
			<td style="width:108px;">Sigma</td>
			<td>A3286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-Alanine</td>
			<td style="width:108px;">Fisher</td>
			<td>S25648&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">p-Aminobenzoic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">A-9878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Biotin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">B-4639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cyanocobalamine (B-12)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">V-2876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Folinate (Ca)&#160;</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">F-7878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacin</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-0761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Niacinamide</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">N-3376</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantetheine</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-2125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pantothenate (Ca)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pteroylglutamic Acid (Folic Acid)</td>
			<td style="width:108px;">ACRCS</td>
			<td style="width:121px;">21663-0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxal 5&#39;-phosphate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-3657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxamine.2HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-9158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridoxine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-6280</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">Riboflavin 5-PO4(Na)</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">R-7774</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Thiamine.HCl</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">DL-6,8-Thioctic Acid</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">T-1395</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:359px;">KH2PO4</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">P-5379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Choline di-acid citrate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">C-2004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">myo-Inositol</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">I-5125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">D-Glucose</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">G-7520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lactalbumin enzymatic hydrolysate</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">L-9010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain Heart Infusion</td>
			<td style="width:108px;">BD</td>
			<td style="width:121px;">211065</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:359px;">Hemin chloride</td>
			<td style="width:108px;">Frontier Scientific</td>
			<td style="width:121px;">H651-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">HEPES, Na salt</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:121px;">H-3784</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Cholesterol</td>
			<td style="width:108px;">J.T. Baker</td>
			<td style="width:121px;">F676-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Non-Essential Amino Acids</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11140-076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Amino Acids Solution</td>
			<td style="width:108px;">Invitrogen</td>
			<td>11130-051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalidixic acid sodium salt</td>
			<td style="width:108px;">Sigma</td>
			<td>N4382</td>
			<td></td>
		</tr>
		<tr height="21">
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culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment

I denne protokol, vi præsentere de nødvendige materialer, og proceduren for modificeret C. E legans tilvænning og Reproduktion medier (mCeHR). Derudover trinene for at udsætte og acclimatizing C. elegans dyrket på E. coli Axeniske flydende medier er beskrevet. Endelig downstream eksperimenter, der anvender aksenisk C. elegans illustrerer fordelene ved denne procedure. Evnen til at analysere og afgøre C. Kravet elegans næringsstof blev illustreret ved at dyrke N2 vildtype orme i axeniske flydende medier med varierende hæm koncentrationer. Denne procedure kan gentages med andre næringsstoffer til at bestemme den optimale koncentration for orm vækst og udvikling, eller for at bestemme de toksikologiske virkninger af medicinsk behandling. Virkningerne af varierede hæm koncentrationer på væksten af ​​vildtype orme blev bestemt gennem kvalitative mikroskopisk observation og ved kvantificering than antal orme, der voksede i hver hæm koncentration. Desuden kan virkningen af ​​forskellige koncentrationer af næringsstoffer, der skal analyseres ved anvendelse af orme, der udtrykker fluorescerende sensorer, der reagerer på ændringer i næringsstoffer af interesse. Desuden blev et stort antal orme let fremstilles til generering af transgene C. elegans hjælp mikropartikelbombardement.

Dyrkning C. elegans i axeniske flydende medium hjælpemidler i fastlæggelsen af næringsstoffer, der kræves af orme, uden indblanding fra sekundære metabolitter produceret af E. coli. Vildtype N2 orme akklimatisere til mCeHR medier inden for tre generationer og vise vækst sammenlignelig for orme dyrket på NGM bakterielle plader. Faktisk er disse orme bliver gravide indenfor 4 dage sammenlignet med 3,5 dage for orme dyrkes på OP50 bakterier. En fordel ved at bruge mCeH...

Watch this Scientific Journal Video about Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment at JoVE.com

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans er normalt dyrkes på faste agarplader eller i flydende kulturer podet med E. coli. At forebygge bakterielle biprodukter fra confounding toksikologiske og ernæringsmæssige undersøgelser vi udnyttet en aksenisk flydende medium, CeHR, at vokse og synkronisere et stort antal orme for en række efterfølgende anvendelser.

Dyrkning<em&gt; Caenorhabditis elegans</em &gt; I Axeniske Liquid Media og fremstilling af transgene Worms ved mikropartikelbombardement

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

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Biology

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Measuring Caenorhabditis elegans Life Span on Solid Media

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 ...

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

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

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 ...

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, ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Dyrkning af human Nasal epithelialceller på Air Liquid grænseflade

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

En screenbar<em&gt; In vivo</em&gt; Assay for mitokondrie modulatorer Brug Transgene Bioluminescent<em&gt; Caenorhabditis elegans</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

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 ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Kombinerede nukleotid og Protein ekstraktioner i Caenorhabditis elegans

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