Name: the production of c. elegans transgenes via recombineering with the galk selectable marker
Uploaded: 2011-01-11T23:58:00
Description: Watch this Scientific Journal Video about The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker at JoVE.com

De auteurs willen graag Lindsey Nash bedanken voor hun hulp met het ontwikkelen van de techniek. Dit werk werd gefinancierd door NIH subsidie ​​AG028977 om ALF, een pilot project subsidie ​​van de universiteit van Pittsburgh OAIC (AG024827), en het zaad fondsen van de universiteit van Pittsburgh.

Overzicht Veel transgenen gebruikt bij het ​​genereren van transgene C. elegans bestaan ​​uit promoter sequenties en misschien een gen cDNA gekloneerd in een van de vectoren die door het laboratorium van dr. Andy Fire 1. Hoewel deze transgenen zijn vaak succesvol met betrekking tot het produceren van een GFP reporter-gen of het uitdrukken van een cDNA in een gewenst patroon, kunnen deze transgenen missen de alternatieve promotors, enhancer-elementen, en 3 &#39;onver...

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De generatie van transgenen uit fosmids biedt het voordeel van behoud van alle van de inheemse promoter elementen, splice-varianten, en 3 &#39;UTR regulerende elementen. Dit kan leiden tot de bouw van een transgen dat is meer een weerspiegeling van de inheemse expressie patroon, of de bouw van een functioneel transgen als andere benaderingen 5 falen. De resulterende transgenen kan dragen een groot aantal epitoop labels waaronder GFP of een TAP tag. De bouw van transgenen in drie s...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>FosmidMAX kit</td>
<td>&nbsp;</td>
<td>Epicentre</td>
<td>FMAX046</td>
<td>&nbsp;</td>
<tr>
<td>GoTaq</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>M7122</td>
<td>&nbsp;</td>
<tr>
<td>MOPS Media</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2120</td>
<td>&nbsp;</td>
<tr>
<td>0.132 M Potassium phosphate solution</td>
<td>&nbsp;</td>
<td>Teknova</td>
<td>M2102</td>
<td>&nbsp;</td>
<tr>
<td>D-galactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G0750</td>
<td>&nbsp;</td>
<tr>
<td>2-deoxygalactose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D4407</td>
<td>&nbsp;</td>
<tr>
<td>Biotin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4639</td>
<td>&nbsp;</td>
<tr>
<td>Leucine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L8000</td>
<td>&nbsp;</td>
<tr>
<td>NH4Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9434</td>
<td>&nbsp;</td>
<tr>
<td>Phusion DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>F-530S</td>
<td>&nbsp;</td>
<tr>
<td>MacConkey agar base</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>281810</td>
<td>&nbsp;</td>
<tr>
<td>Arabinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3131</td>
<td>&nbsp;</td>
<tr>
<td>Chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1919</td>
<td>&nbsp;</td>
<tr>
<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5136</td>
<td>&nbsp;</td>
<tr>
<td>Potassium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5655</td>
<td>&nbsp;</td>
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5886</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G2025</td>
<td>&nbsp;</td>
<tr>
<td>Bacto Agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>214010</td>
<td>&nbsp;</td>		</tbody>
		</table>
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the production of c. elegans transgenes via recombineering with the galk selectable marker

Het creëren van transgene dieren wordt op grote schaal gebruikt in C. elegans onderzoek met inbegrip van het gebruik van GFP fusie-eiwitten voor de regulering en expressie patroon van de genen van belang of generatie van de tandem affiniteitszuivering (TAP) gelabelde versies van specifieke genen om hun zuivering te vergemakkelijken studie. Typisch transgenen worden gegenereerd door het plaatsen van een promotor stroomopwaarts van een GFP reporter-gen of cDNA van belang, en dit vaak levert een vertegenwoordiger expressiepatroon. Echter, kritische elementen van de genregulatie, zoals de controle-elementen in onvertaald de drie &#39;regio of alternatieve promotors, kan gemist worden door deze aanpak. Verder slechts een splice variant kan doorgaans worden bestudeerd langs deze weg. In tegenstelling tot het gebruik van de worm genomisch DNA gedragen door fosmid DNA-klonen bevat waarschijnlijk de meeste, zo niet alle elementen die betrokken zijn bij genregulatie in vivo waarin de groter vermogen om de echte uitdrukking patroon en de timing vast te leggen vergunningen. Ter vergemakkelijking van de generatie van transgenen via fosmid DNA, beschrijven we een E. coli op basis van recombineering procedure om GFP, een TAP-tag, of andere sequenties van belang te voegen in elke gewenste locatie in het gen. De procedure maakt gebruik van de galK gen als de selectie marker voor zowel de positieve en negatieve selectie stappen in recombineering wat resulteert in het verkrijgen van de gewenste wijziging met een hoge efficiëntie. Verder, plasmiden die de galK gen geflankeerd door homologie armen tot veelgebruikte GFP en TAP fusie-genen zijn beschikbaar die de kosten van de oligos te verminderen met 50% bij het ​​genereren van een GFP of TAP fusie-eiwit. Deze plasmiden gebruik maken van de R6K replicatieoorsprong die de noodzaak voor uitgebreide PCR product zuivering uitsluit. Tot slot hebben we ook aantonen dat er een techniek om de unc-119 marker te integreren op de fosmid ruggengraat die het mogelijk maakt de fosmid direct worden geïnjecteerd of gebombardeerd in de wormen om transgene dieren te genereren. Deze video toont de procedures die betrokken zijn bij het genereren van een transgen via recombineering met deze methode.

Watch this Scientific Journal Video about The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker at JoVE.com

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

De mogelijkheid om transgenen te produceren voor<em&gt; Caenorhabditis elegans</em&gt; Gebruik van genomisch DNA gedragen door fosmids is vooral aantrekkelijk als alle van de inheemse regulerende elementen zijn behouden. Beschreven, is een eenvoudige en deugdelijke procedure voor de productie van transgenen via recombineering met de<em&gt; GalK</em&gt; Selecteerbare merker.

De productie van C. elegans Transgenen via Recombineering met de GalK Selecteerbare marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

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The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

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High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

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Het creëren van Defined Gasvormige omgevingen om de gevolgen van hypoxie Studie naar<em&gt; C. elegans</em

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Met behulp van RNA-gemedieerde interferentie voerstrategie om te screenen op genen die betrokken zijn Lichaam Grootte verordening in de nematode<em&gt; C. elegans</em

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Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

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Een snelle protocol voor de integratie Extrachromosomaal Arrays Met High Transmission Rate in de<em&gt; C. elegans</em&gt; Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...