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 arbeidet ble støttet av National Institutes of HealthGrants DK85035 og DK074797 (IH).

En. Worm Stammer <ol><li> Skaffe C. elegans villtype Bristol N2 stammer fra Caenorhabditis Genetics Center (CGC) (http://www.cbs.umn.edu/cgc) og vedlikeholde dem på NGM plater seeded med E. coli stamme OP50 7. Merk: Transgenic ormen stammer IQ6011 (Phrg-1 :: GFP) utnyttet ble generert som tidligere beskrevet 11. IQ6011 kan fås fra tilsvarende forfatteren. </li></ol> 2. Utarbeidelse av Modified C. elegans Habita...

<ol>
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I denne protokollen presenterer vi en modifisert axenic flytende medier mCeHR som gir mulighet for rask C. elegans generasjon med produksjon av et stort antall ormer. Dette mediet viser flere fordeler som ormene er dyrket uten forurensende E. coli eller bakteriell biprodukter og kan utnyttes i ernæringsmessige og toksikologiske studier. Bruken av E. coli eller andre bakterier i slike studier har flere ulemper. For eksempel, kan veksten av bakteriene endres under forskjellige betingelser, og d...

Jordsmonnet nematode, Caenorhabditis elegans, er en kraftig modell organisme som brukes i en rekke studier fra genetikk til toksikologi. Som et resultat av dens 1 mm størrelse, hurtig generasjonstid på fire dager, enkle dyrking, og store avkoms tall, har disse nematoder blitt benyttet i en rekke genetiske og farmakologiske skjermer 1, 2. Forskere dra nytte av denne ormen å identifisere molekyler og trasé bevart i virveldyr systemer. Disse banene inkluderer celledød signaler, veier av aldring og metabolisme, og nervesystemet 3-6. I tillegg, gjennomsiktigheten C. elegans tillater generering av transgene linjer ved hjelp av fluorescerende protein journalister, noe som kan være direkte visualisert å analysere genuttrykk mønstre og protein lokalisering. I mange studier denne nematode er dyrket på en solid agar-baserte overflaten ved hjelp av nematode vekstmedium (NGM) plater eller i lVæsken kulturer seeded med Escherichia coli som matkilde 7,8. Disse bakterielle mat kilder kan forvirre biokjemiske og toksikologiske studier med forstyrrelser fra bakteriell biprodukter som påvirker tolkningen av resultatene. For å unngå disse sammensatte virkninger, C. elegans kan dyrkes i en axenic flytende medier som er blottet for bakterier som matkilde. Ved hjelp av dette mediet, vi får dyrket millioner av svært synkroniserte ormer for mange standard C. elegans protokoller inkludert microarray analyse av differensielt regulerte gener i C. elegans utsatt for ulike heme konsentrasjoner, og produksjon av transgene ormer som bruker genet bombardement. Dette mediet er kjemisk definerte og endret fra en original oppskrift formulert av Dr. Eric Clegg ni. Ved hjelp av denne mCeHR medier, har vi lykkes identifiserte gener involvert i heme homeostase, referert til som heme responsive gener (HRG e) 10, som villehar ikke vært mulig i vanlige vekstbetingelser som utnytter NGM agarplater podet med E. coli. I denne protokoll beskrives fremgangsmåten for innføring og opprettholdelse C. elegans dyrket på E. coli til axenic mCeHR og benytte denne metode for å oppnå et stort antall ormer for fremstilling av transgene C. elegans linjer ved hjelp av mikropartikkel bombardement. Vi presenterer også studier som viser nytten av å bruke axenic media for å bestemme ernæringsmessige krav om C. elegans hjelp heme som et eksempel. Disse studiene viser at ved bruk av mCeHR media har en rask vekst av et stort antall C. elegans for mange nedstrøms programmer benyttes av 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">
			<td height="21" style="height:21px;">Tetracycline Hydrochloride</td>
			<td style="width:108px;">MP Biomedicals</td>
			<td>2194542</td>
			<td></td>
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culturing caenorhabditis elegans in axenic liquid media and creation of transgenic worms by microparticle bombardment

I denne protokollen, presenterer vi de nødvendige materialer, og prosedyren for å lage modifiserte C. E legans Tilvenning og reproduksjon media (mCeHR). I tillegg, er fremgangsmåten for å utsette og acclimatizing C. elegans dyrket på E. coli til axenic flytende medier er beskrevet. Endelig, nedstrøms eksperimenter som utnytter axenic C. elegans illustrere fordelene ved denne prosedyren. Evnen til å analysere og finne C. elegans næringsstoff kravet ble illustrert ved å dyrke N2 vill type ormer i axenic flytende medier med varierende heme konsentrasjoner. Denne prosedyren kan replikeres med andre næringsstoffer for å fastslå den optimale konsentrasjonen for orm vekst og utvikling, eller, for å bestemme de toksikologiske effektene av medikamentell behandling. Virkningene av varierende heme konsentrasjoner på veksten av vill-type ormer ble bestemt ved kvalitativ mikroskopisk observasjon og ved kvantifisering tHan antall ormer som vokste i hvert heme konsentrasjon. I tillegg kan effekten av varierende næringskonsentrasjoner analyseres ved anvendelse av ormer som uttrykker fluorescerende sensorer som reagerer på endringer i det næringsstoffet er av interesse. Videre er et stort antall ormer ble lett fremstilt for generering av transgene C. elegans ved hjelp av mikropartikkel bombardement.

Dyrking C. elegans i axenic flytende medium hjelpemidler i fastsettelse av næringsstoffer som kreves av ormer, uten innblanding fra sekundære metabolitter produsert av E. coli. Villtype N2 ormer akklimatisere til mCeHR media i løpet av tre generasjoner og vise vekst sammenlignes med ormer dyrket på NGM bakterielle plater. Faktisk disse ormer bli gravid løpet av 4 dager, sammenlignet med 3,5 dager for ormer dyrket på OP50 bakterier. En fordel med å bruke mCeHR ble set...

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 vanligvis dyrket på fast agar plater eller i flytende kulturer seeded med E. coli. For å hindre bakterie biprodukter fra blander sammen toksikologiske og ernæringsmessige undersøkelser, benyttet vi en axenic flytende medium, CeHR, for å vokse, og synkronisere et stort antall ormer for en rekke nedstrøms applikasjoner.

Dyrking<em&gt; Caenorhabditis elegans</em &gt; I Axenic Flytende Media og Opprettelse av Transgene Worms av mikropartikkel bombardementet

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

Grunnleggende<em&gt; Caenorhabditis elegans</em&gt; Metoder: Synkronisering og observasjon

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 Utvinning 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

Dyrking av menneskelig Nasal Epitelceller på Air Flytende Interface

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 silbart<em&gt; I Vivo</em&gt; Analyse for mitokondrie modulatorer Bruke Transgenic 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

dyrking av<em&gt; Caenorhabditis elegans</em&gt; I tre dimensjoner 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

Kombinert Nucleotide og Protein utdrag 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 ...