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

Os autores gostariam de agradecer a Lindsey Nash para ajudar a desenvolver a técnica. Este trabalho foi financiado pelo NIH conceder AG028977 a ALF, uma bolsa de projeto piloto da Universidade de Pittsburgh OAIC (AG024827), e fundos de sementes da Universidade de Pittsburgh.

Visão global Transgenes muitos usados ​​na geração de transgênicos C. elegans consistem em seqüências de promotor e, talvez, um gene cDNA clonado em um dos vetores gerados pelo laboratório do Dr. Andy Fogo 1. Embora esses transgenes são frequentemente bem sucedidos no que diz respeito à elaboração de um gene repórter GFP ou expressar um cDNA em um padrão desejado, esses transgenes pode faltar os promotores alternativos, os elementos enhancer, e 3 &#39;não ...

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A geração de transgenes de fosmids oferece o benefício de manter todos os elementos promotor nativo, splice variantes, e três elementos &#39;UTR de regulamentação. Isso pode levar à construção de um transgene que é mais um reflexo do padrão de expressão nativa, ou a construção de um transgene funcional quando outras abordagens não 5. Os transgenes resultante pode carregar uma variedade de etiquetas de epítopo incluindo GFP ou um tag TAP. A construção de transgene...

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		<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>
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the production of c. elegans transgenes via recombineering with the galk selectable marker

A criação de animais transgênicos é amplamente utilizada em C. elegans pesquisa, incluindo o uso de proteínas de fusão GFP para estudar a regulação e padrão de expressão de genes de interesse ou de geração de conjunto de purificação de afinidade (TAP) com a tag versões de genes específicos para facilitar a sua purificação. Tipicamente transgenes são gerados pela colocação de um promotor a montante de um gene repórter GFP ou cDNA de interesse, e isso muitas vezes produz um padrão de expressão representativa. No entanto, elementos críticos da regulação de genes, como elementos de controle na região não traduzida 3 &#39;ou promotores alternativos, podem ser perdidos por esta abordagem. Mais apenas uma variante única emenda pode ser geralmente estudado por este meio. Em contraste, o uso de DNA genômico verme transportado por clones DNA fosmid provavelmente inclui a maioria se não todos os elementos envolvidos na regulação de genes in vivo que permite a maior capacidade de capturar o padrão de expressão genuína e timing. Para facilitar a geração de transgenes usando DNA fosmid, descrevemos um E. coli baseado recombineering procedimento para inserir GFP, a TAP tag, ou outras seqüências de interesse em qualquer local do gene. O procedimento utiliza o gene galK como o marcador de seleção para as duas etapas de seleção positiva e negativa em recombineering o que resulta na obtenção da modificação desejada com alta eficiência. Além disso, plasmídeos contendo o gene galK ladeado por braços homologia com GFP comumente usados ​​e TAP genes de fusão estão disponíveis que reduzem o custo de oligos em 50% ao gerar um GFP, ou proteína de fusão TAP. Estes plasmídeos use a origem de replicação R6K que impede a necessidade de purificação extensa produto da PCR. Finalmente, também demonstram uma técnica para integrar o marcador unc-119 para o backbone fosmid que permite que o fosmid a ser injetado diretamente ou bombardeados em vermes para gerar animais transgênicos. Este vídeo demonstra os procedimentos envolvidos na geração de um transgene através recombineering utilizando este método.

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

A capacidade de produzir transgenes para<em&gt; Caenorhabditis elegans</em&gt; Usando DNA genômico realizado por fosmids é particularmente atraente como todos os elementos nativos reguladoras são retidos. Descrito é um procedimento simples e robusto para a produção de transgenes via recombineering com o<em&gt; GalK</em&gt; Marcador selecionável.

Produção de C. elegans Transgenes via Recombineering com o GalK Marcador selecionável

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