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

Författarna vill tacka Lindsey Nash för att få hjälp med att utveckla tekniken. Detta arbete har finansierats av NIH bidrag AG028977 till ALF, ett pilotprojekt bidrag från University of Pittsburgh OAIC (AG024827) och medel frö från University of Pittsburgh.

Översikt Många transgener används i produktionen av transgena C. elegans består av promotorn sekvenser och kanske en gen cDNA klonad till en av de vektorer som genereras av lab av Dr Andy Fire 1. Även om dessa transgener är ofta framgångsrika när det gäller att producera en gen GFP reporter eller uttrycker ett cDNA i önskad mönster, kan dessa transgener saknar alternativa initiativtagare, förstärkare element och 3 &quot;oöversatta regionen (UTR) element som sp...

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Den generation av transgener från fosmids erbjuder förmånen att behålla alla de infödda promotorn element, varianter skarva och 3 &#39;UTR reglerande element. Detta kan leda till byggandet av en transgen som är mer reflekterande av de infödda uttryck mönster, eller byggandet av en funktionell transgen när andra metoder misslyckas 5. Den resulterande transgener kan bära en mängd olika epitop taggar inklusive GFP eller en TAP tagg. Byggandet av transgener omfattat tre ste...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<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>
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<td>Biotin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4639</td>
<td>&nbsp;</td>
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<td>Leucine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L8000</td>
<td>&nbsp;</td>
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<td>NH4Cl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9434</td>
<td>&nbsp;</td>
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<td>Phusion DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>F-530S</td>
<td>&nbsp;</td>
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<td>MacConkey agar base</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>281810</td>
<td>&nbsp;</td>
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<td>Arabinose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3131</td>
<td>&nbsp;</td>
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<td>Chloramphenicol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C1919</td>
<td>&nbsp;</td>
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<td>Sodium phosphate dibasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5136</td>
<td>&nbsp;</td>
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<td>Potassium phosphate monobasic</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5655</td>
<td>&nbsp;</td>
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<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S5886</td>
<td>&nbsp;</td>
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<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>
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the production of c. elegans transgenes via recombineering with the galk selectable marker

Skapandet av transgena djur är allmänt används i C. elegans forskning inklusive användningen av GFP fusionsproteiner att studera reglering och uttryck mönster av gener av intresse eller generering av tandem affinitet rening (TAP) taggade versioner av specifika gener för att underlätta deras rening. Normalt transgener genereras genom att placera en främjare före ett GFP reporter gen eller cDNA av intresse, och detta ger ofta ett representativt uttryck mönster. Men, kritiska delar av genreglering, såsom kontroll element i 3 &#39;oöversatta regionen eller initiativtagare alternativ skulle kunna missas av denna strategi. Ytterligare en enda skarv variant kan oftast studeras på detta sätt. Däremot innefattar användning av masken arvsmassans DNA bärs av fosmid DNA-kloner sannolikt de flesta om inte alla element som ingår i genreglering in vivo som tillåter större förmåga att fånga det äkta uttrycket mönster och timing. För att underlätta skapandet av transgener använda fosmid DNA, beskriver vi ett E. coli baserade recombineering för att infoga GFP, en TAP-tagg eller andra sekvenser av intresse i någon plats i genen. Förfarandet använder galK genen som val markör för både positiva och negativa val steg i recombineering vilket resulterar i att få den önskade ändringen med hög verkningsgrad. Vidare plasmider innehåller galK genen flankerad av homologi vapen till vanliga GFP och TAP fusionsgener finns tillgängliga som minskar kostnaderna för oligos med 50% när du genererar en GFP eller TAP fusionsprotein. Dessa plasmider använda R6K replikering ursprung som utesluter behovet av omfattande PCR-produkt rening. Slutligen visar vi också en teknik för att integrera UNC-119 markör på den fosmid ryggrad som gör att fosmid vara direkt injiceras eller bombarderas med maskar för att skapa transgena djur. Denna video demonstrerar förfarandet i samband generera en transgen via recombineering med denna metod.

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

Förmågan att producera transgener för<em&gt; Caenorhabditis elegans</em&gt; Använda genomisk DNA bärs av fosmids är särskilt attraktiva som alla infödda reglerande element behålls. Beskrivs är ett enkelt och stabilt förfarande för framställning av transgener via recombineering med<em&gt; GalK</em&gt; Markör.

Produktionen av C. elegans Transgener via Recombineering med GalK Markör

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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Skapa Definierade Gasformiga miljöer för att studera effekterna av Hypoxi på<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

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Använda RNA-medierad strategi Interference Matning att screena för gener involverade i kroppsstorlek förordningen i rundmasken<em&gt; C. elegans</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

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C. elegans kemotaxianalys

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

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