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

著者は、技術の開発の手助けのためにリンジーナッシュに感謝します。この作品は、ALF、ピッツバーグ大学からピッツバーグOAIC（AG024827）、およびシード資金の大学からのパイロットプロジェクトの助成にNIHの助成金AG028977によって賄われていた。

概要トランスジェニックC.の世代で使用されている多くの組換え遺伝子虫は、プロモーター配列と博士アンディ火1のラボで生成されたベクトルの一つにクローニングされたcDNAを、おそらく遺伝子で構成されています。これらの組換え遺伝子が頻繁にGFPレポーター遺伝子を製造または所望のパターンでcDNAを発現に関してで成功している一方で、これら?...

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fosmidsから導入遺伝子の世代は、ネイティブなプロモーターエレメント、スプライシング変異体、および3&#39;UTR調節要素のすべてを保持することの利点を提供しています。これは、ネイティブの発現パターン、または他のアプローチは5失敗した機能的な導入遺伝子の構造をより反映し、導入遺伝子の構築につながることができます。その結果導入遺伝子は、GFPまたはTAPのタグを含む?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<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>
		</div>

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

トランスジェニック動物の作成 ​​が広く、Cで利用されている興味またはタンデムアフィニティー精製の ​​世代の遺伝子の調節と発現パターンを研究するためにGFPの融合タンパク質の使用を含む線虫の研究（TAP）その精製を容易にするために特定の遺伝子のタグ付きバージョン。一般的に導入遺伝子はGFPレポーター遺伝子または目的のcDNAの上流にプロモーターを配置することで生成され、これはしばしば代表的発現パターンを生成します。しかし、このような3&#39;非翻訳領域または別のプロモーターの制御要素として遺伝子調節の重要な要素は、このアプローチによって検出し損ねる可能性がある。さらに、単一のスプライスバリアントは、通常はこの方法で研究することができる。対照的に、フォスミドDNAクローンによって運ばの使用ワームのゲノムDNAは、おそらくほとんどの場合ではない本物の発現パターンとタイミングをキャプチャする大きな能力を許可する生体内で遺伝子の調節に関与するすべての要素が含まれています。フォスミドDNAを用いて導入遺伝子の生成を容易にするために、我々はE.を説明大腸菌は遺伝子の任意の場所にGFP、TAP -タグ、または興味の他の配列を挿入するrecombineering手順をベースに。手順は、高効率で目的の変更を取得、その結果recombineeringで正と負の選択の手順の両方のための選択マーカーとしてgalK遺伝子を使用しています。さらに、一般的に使用されるGFPとTAPの融合遺伝子に相同性ア ​​ームに挟まれたgalKの遺伝子を含むプラスミドは、GFPまたはTAP融合タンパク質を生成する際に50％のオリゴのコストを減少する可能です。これらのプラスミドは、豊富なPCR産物の精製の必要性を排除R6Kの複製起点を使います。最後に、我々はまた、フォスミドを直接注入またはトランスジェニック動物を生成するためにワームに砲撃できるようにするフォスミドバックボーンへのUNC - 119マーカーを統合する手法を示す。このビデオでは、このメソッドを使用してrecombineeringを経由して導入遺伝子の生成に関わる手順を示しています。

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

のために導入遺伝子を産生する能力<em&gt;線虫（Caenorhabditis elegans）</em&gt;ネイティブ調節要素のすべてが保持されるようにfosmidsによって運ばれるゲノムDNAを使うことが特に魅力的です。説明でrecombineering経由導入遺伝子の生産のためのシンプルで堅牢な手続きです。<em&gt; galK</em&gt;選択可能なマーカー。

の生産 C.エレガンス</emとRecombineering経由>トランスジーン galK選択可能なマーカー

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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Biology

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

Generation of Stable Transgenic C. elegans Using Microinjection

マイクロインジェクションを使用して安定したトランスジェニック線虫の世代

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

潜在的なパーキンソン病の遺伝子を検証するための線虫のドーパミンニューロンの変性のアッセイのアプリケーション

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

解離性とC. elegansの神経筋接合部から録音する

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

A Protocol for the Production of KLRG1 Tetramer

KLRG1テトラマーの生産のためのプロトコル

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

計測線虫（Caenorhabditis elegans）</em96穴マイクロタイタープレートの>寿命

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

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

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

に低酸素の影響を検討するために定義され気体環境を作成する<em&gt; C.エレガンス</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

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

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた<em&gt; Cエレガンス</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 ...

C. elegans Chemotaxis Assay

C. elegans Chemotaxis Assay

C. elegansの走化性アッセイ

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

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

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

の生産 C.エレガンストランスジーン galK選択可能なマーカー