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

Questo lavoro è stato sostenuto dal National Institutes of HealthGrants DK85035 e DK074797 (IH).

1. Ceppi Worm <ol><li> Ottenere C. elegans tipo selvatico ceppi Bristol N2 dal Caenorhabditis Genetics Center (CGC) (http://www.cbs.umn.edu/cgc) e li mantengono su piastre NGM seminato con E. coli ceppo OP50 7. Nota: verme transgenico ceppi IQ6011 (Phrg-1 :: GFP) utilizzati sono stati generati come descritto in precedenza 11. IQ6011 può essere richiesto all&#39;autore corrispondente. </li></ol> 2. Preparazione di Modifie...

<ol>
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In questo protocollo vi presentiamo un axenic mCeHR mezzi liquidi modificato che consente una rapida C. generazione elegans con produzione di un gran numero di vermi. Questo supporto mostra diversi vantaggi come i vermi sono coltivate senza contaminare E. coli o sottoprodotti batterici e possono essere sfruttati in studi nutrizionali e tossicologici. L&#39;uso di E. coli o altri batteri in detti studi ha diversi inconvenienti. Ad esempio, la crescita dei batteri può cambiare in varie...

Il nematode del suolo, Caenorhabditis elegans, è un potente organismo modello utilizzato in numerosi studi dalla genetica alla tossicologia. Come risultato della sua dimensione 1 mm tempo di generazione rapida di quattro giorni facilità di coltivazione, e un gran numero di progenie, questi nematodi sono stati utilizzati in un numero di schermi genetici e farmacologici 1, 2. Ricercatori approfittare di questo worm per identificare molecole e percorsi conservati nei sistemi di vertebrati. Questi percorsi sono segnali di morte cellulare, percorsi di invecchiamento e del metabolismo, e il sistema nervoso 3-6. Inoltre, la trasparenza di C. elegans consente la generazione di linee transgeniche utilizzando reporter proteine ​​fluorescenti, che possono essere visualizzati direttamente per analizzare i modelli di espressione genica e localizzazione delle proteine. In molti studi questo nematode è coltivato su una superficie a base di agar-solido utilizzando nematode mezzo di crescita (NGM) piastre o in lculture iquid seminati con Escherichia coli come fonte di cibo 7,8. Queste fonti di cibo batteriche possono confondere studi biochimici e tossicologici con interferenza da batteri sottoprodotti che influenzano l&#39;interpretazione dei risultati. Per evitare questi effetti compounding, C. elegans possono essere coltivate in un mezzo liquido axeniche che è privo di batteri come fonte di cibo. Usando questo supporto, abbiamo coltivato con successo milioni di vermi altamente sincronizzati per molti di serie C. protocolli elegans, compresa l&#39;analisi microarray di geni differenzialmente regolati in C. elegans esposto a diverse concentrazioni eme, e produzione di vermi transgenici utilizzando gene bombardamento. Questo supporto è chimicamente definita e modificata da una ricetta originale formulata dal Dr. Eric Clegg 9. Usando questo supporto mCeHR, abbiamo identificato con successo geni coinvolti nella eme omeostasi, riferiti ai geni come eme responsivi (HRG s) 10, che avrebbenon sarebbe stato possibile in condizioni di crescita regolari che utilizzano piastre di agar NGM seminato con E. coli. In questo protocollo si descrive la procedura per l&#39;introduzione e il mantenimento C. elegans cresciuti su E. coli per la axenic mCeHR e utilizzare questo metodo per ottenere un gran numero di vermi per produrre transgenico C. Linee elegans utilizzando microparticelle bombardamento. Abbiamo presenti anche studi che dimostrano l&#39;utilità di utilizzare supporti axeniche per la determinazione del fabbisogno nutrizionale di C. elegans utilizzando eme come esempio. Questi studi mostrano che utilizzando mCeHR supporti consente la rapida crescita di un gran numero di C. elegans per molte applicazioni a valle utilizzate dai ricercatori worm.

<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

In questo protocollo, presentiamo i materiali necessari, e la procedura per fare C modificati. Legans e assuefazione e della Riproduzione media (mCeHR). Inoltre, la procedura per l&#39;esposizione e acclimatazione C. elegans cresciuti su E. coli a axeniche mezzi liquidi sono descritti. Infine, gli esperimenti a valle che utilizzano axenic C. elegans illustrano i benefici di questa procedura. La capacità di analizzare e determinare C. fabbisogno nutritivo elegans è stato illustrato da una crescente N2 wild type vermi in mezzi liquidi axeniche con concentrazioni variabili eme. Questa procedura può essere replicata con altri nutrienti per determinare la concentrazione ottimale per la crescita e lo sviluppo verme o, per determinare gli effetti tossicologici dei trattamenti farmacologici. Gli effetti di varie concentrazioni eme sulla crescita di tipo selvaggio vermi sono stati determinati mediante osservazione microscopica qualitativa e quantificazione tegli numero di worm che crescevano in ciascuna concentrazione eme. Inoltre, l&#39;effetto di varie concentrazioni di nutrienti può essere saggiata utilizzando vermi che esprimono sensori fluorescenti che rispondono alle variazioni del nutriente di interesse. Inoltre, un gran numero di vermi erano facilmente prodotti per la generazione di transgenici C. elegans utilizzando microparticelle bombardamento.

Coltura C. elegans in axeniche aiuti mezzo liquido nella determinazione di sostanze nutritive che sono richiesti dai vermi, senza interferenze da parte di metaboliti secondari prodotti da E. coli. Worm di tipo selvatico N2 acclimatare i media mCeHR entro tre generazioni e mostrano una crescita paragonabile a vermi cresciuti su piastre NGM batteriche. Infatti, questi vermi diventano gravide entro 4 giorni, rispetto a 3,5 giorni per i vermi coltivati ​​sui batteri OP50. U...

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 è di solito coltivate su piastre di agar solidi o liquidi in colture seminate con E. coli. Per evitare sottoprodotti batterici da confondere studi tossicologici e nutrizionali, abbiamo utilizzato un mezzo liquido axenic, CeHR, crescere e sincronizzare un gran numero di vermi per una gamma di applicazioni a valle.

Culturing<em&gt; Caenorhabditis elegans</em &gt; In axeniche Liquid Media e la creazione di transgeniche Worms da microparticelle Bombardamento

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

Research

JoVE Journal

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

Misura Caenorhabditis elegans Life Span su terreni solidi

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

Ricerca

Biologia

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

Generazione di transgenici C. elegans Di Trasformazione Biolistic

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

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Time-lapse Microscopy of Early Embryogenesis in 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

Basic Caenorhabditis elegans Methods: Synchronization and Observation

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

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

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, ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

Coltura di cellule umane nasale epiteliali presso l&#39;Air Interface Liquid

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

Un Screenable<em&gt; In Vivo</em&gt; Saggio per mitocondriale modulatori Uso 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

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

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

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 ...