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。

<ol>
	<li>Mello, C., Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+transformation.">DNA transformation.</a> Methods Cell Biol. 48, 451-482 (1995).</li><li>Zhang, Y., Nash, L., Fisher, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified,+robust,+and+streamlined+procedure+for+the+production+of+C.+elegans+transgenes+via+recombineering.">A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.</a> BMC Dev Biol. 8, 119-119 (2008).</li><li>Antebi, A., Yeh, W. H., Tait, D., Hedgecock, E. M., Riddle, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=daf-12+encodes+a+nuclear+receptor+that+regulates+the+dauer+diapause+and+developmental+age+in+C.+elegans.">daf-12 encodes a nuclear receptor that regulates the dauer diapause and developmental age in C. elegans.</a> Genes and Development. 14, 1512-1527 (2000).</li><li>Snow, M. I., Larsen, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+expression+of+daf-12:+a+nuclear+hormone+receptor+with+three+isoforms+that+are+involved+in+development+and+aging+in+Caenorhabditis+elegans.">Structure and expression of daf-12: a nuclear hormone receptor with three isoforms that are involved in development and aging in Caenorhabditis elegans.</a> Biochim. Biophys. Acta. 1494, 104-116 (2000).</li><li>Fisher, A. L., Page, K. E., Lithgow, G. J., Nash, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+K10C2.4+Gene+Encodes+a+Member+of+the+Fumarylacetoacetate+Hydrolase+Family.+A+CAENORHABDITIS+ELEGANS+MODEL+OF+TYPE+I+TYROSINEMIA.">The Caenorhabditis elegans K10C2.4 Gene Encodes a Member of the Fumarylacetoacetate Hydrolase Family. A CAENORHABDITIS ELEGANS MODEL OF TYPE I TYROSINEMIA.</a> J Biol.Chem. 283, 9127-9135 (2008).</li><li>Wightman, B., Ha, I., Ruvkun, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranscriptional+regulation+of+the+heterochronic+gene+lin-14+by+lin-4+mediates+temporal+pattern+formation+in+C.+elegans.">Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans.</a> Cell. 75, 855-862 (1993).</li><li>Lehrbach, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIN-28+and+the+poly(U)+polymerase+PUP-2+regulate+let-7+microRNA+processing+in+Caenorhabditis+elegans.">LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans.</a> Nat Struct Mol Biol. 16, 1016-1020 (2009).</li><li>Tursun, B., Cochella, L., Carrera, I., Hobert, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+toolkit+and+robust+pipeline+for+the+generation+of+fosmid-based+reporter+genes+in+C.+elegans.">A toolkit and robust pipeline for the generation of fosmid-based reporter genes in C. elegans.</a> PLoS One. 4, e4625-e4625 (2009).</li><li>Bamps, S., Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+gene+expression+pattern+analysis,+in+situ,+in+Caenorhabditis+elegans.">Large-scale gene expression pattern analysis, in situ, in Caenorhabditis elegans.</a> Brief. Funct. Genomic. Proteomic. , (2008).</li><li>Dolphin, C. T., Hope, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+reporter+fusion+genes+generated+by+seamless+modification+of+large+genomic+DNA+clones.">Caenorhabditis elegans reporter fusion genes generated by seamless modification of large genomic DNA clones.</a> Nucleic Acids Res. 34, e72-e72 (2006).</li><li>Sarov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recombineering+pipeline+for+functional+genomics+applied+to+Caenorhabditis+elegans.">A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.</a> Nat. Methods. 3, 839-844 (2006).</li><li>Yang, X. W., Model, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homologous+recombination+based+modification+in+Escherichia+coli+and+germline+transmission+in+transgenic+mice+of+a+bacterial+artificial+chromosome.">Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome.</a> Nat Biotechnol. 15, 859-865 (1997).</li><li>Court, D. L., Sawitzke, J. A., Thomason, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+using+homologous+recombination.">Genetic engineering using homologous recombination.</a> Annu.Rev.Genet. 36, 361-388 (2002).</li><li>Westenberg, M., Bamps, S., Soedling, H., Hope, I. A., Dolphin, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+MW005:+lambda+Red-mediated+recombineering+and+copy-number+induction+of+oriV-equipped+constructs+in+a+single+host.">Escherichia coli MW005: lambda Red-mediated recombineering and copy-number induction of oriV-equipped constructs in a single host.</a> BMC Biotechnol. 10, 27-27 (2010).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36-e36 (2005).</li><li>Penfold, R. J., Pemberton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+suicide+vector+for+construction+of+chromosomal+insertion+mutations+in+bacteria.">An improved suicide vector for construction of chromosomal insertion mutations in bacteria.</a> Gene. 118, 145-146 (1992).</li><li>Praitis, V., Casey, E., Collar, D., Austin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creation+of+low-copy+integrated+transgenic+lines+in+Caenorhabditis+elegans.">Creation of low-copy integrated transgenic lines in Caenorhabditis elegans.</a> Genetics. 157, 1217-1226 (2001).</li><li>Puig, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tandem+affinity+purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The tandem affinity purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 (2001).</li><li>Rigaut, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterization+and+proteome+exploration.">A generic protein purification method for protein complex characterization and proteome exploration.</a> Nat.Biotechnol. 17, 1030-1032 (1999).</li><li>Achilleos, A., Wehman, A. M., Nance, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAR-3+mediates+the+initial+clustering+and+apical+localization+of+junction+and+polarity+proteins+during+C.+elegans+intestinal+epithelial+cell+polarization.">PAR-3 mediates the initial clustering and apical localization of junction and polarity proteins during C. elegans intestinal epithelial cell polarization.</a> Development. 137, 1833-1842 (2010).</li><li>Maduro, M., Pilgrim, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+cloning+of+unc-119,+a+gene+expressed+in+the+Caenorhabditis+elegans+nervous+system.">Identification and cloning of unc-119, a gene expressed in the Caenorhabditis elegans nervous system.</a> Genetics. 141, 977-988 (1995).</li></ol>

利害の衝突は宣言されません。

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のタグを含むエピトープタグの様々を運ぶことができます。 導入遺伝子の構造は、すべてのSW106細菌の染色15で実施される3つの手順を関与。最初に、galKの遺伝子は、標的のフォスミドと相補的な相同性ア ​​ームに挟まれたPCR産物運ぶgalKでrecombineeringを介して変更するフォスミドで目的のサイトに統合されています。我々が開発した改良は、FLAG - GFP或いはFosmid変更の第1及び第2段階で使用されるとオリゴのコストを下げることにオリゴの単 ​​一のセットを可能にgalKカセットにTAPタグのいずれかの相同性領域の統合です。さらに簡素化は、SW106菌株では複製できないと親のベクトルを消化する必要がなくなりますR6Kベースのベクターの使用です。第二、galKカセットは、フォスミド上に目的の遺伝子と融合しているようなGFPやTAPなどのタグに置き換えられます。このステップは非常に堅牢であり、多くの適切な変更fosmidsは日常的に得られる。最後に、トランスジェニックワームの世代のためのUNC - 119選択可能なマーカーは、フォスミドに追加されます。これは、シスのマーカーを作成し、直接変更フォスミドにリンク。 UNC - 119選択可能なマーカーは、幅広く、マイクロインジェクションし、遺伝子銃砲撃17,21の両方と互換性があります。砲撃は、トランスジェニック系統のかなりの数が、単一の臨床試験によって生成することができ、経験の浅い担当者がトランスジェニック動物を作成できるので特に便利です。 このプロトコルの重要な側面は、SW106コンピテントセルの調製時のヒートショック手順振盪水浴インキュベーターの使用です。我々は空気インキュベーターの使用はは° Cにもシェーカーで30分（図示せず）の後に42に細菌培養を加熱することができないことがわかった。我々はまた、次善の結果（図示せず）と水浴中の細菌を浸漬試みた。振とう水浴は、すでに企業の数でない場合は研究室で利用できるから使用購入することができます。 GFPまたはエピトープタグの挿入を超えて、我々はまた、fosmids（A.ファーガソンとA.フィッシャー、未発表）で運ばワームの遺伝子の部位特異的変異誘発のためにこのテクニックを使用し始めている。これは、galK遺伝子を置き換えるために、サイトの上流と下流50bpの挟まれた変更を含む一本鎖オリゴを使用することによって達成された。遺伝子のノックアウト変異体の使用と相まってフォスミド変異導入の組み合わせは、特定の遺伝子のアイソフォーム、ドメイン、または決定する特定のアミノ酸の生体機能に可能性があります。それは追加のエピトープのタグを挿入したり、タグ付けされた構造上の変異導入を行うために変更の追加のラウンドを行うことができるので、さらに我々のプロトコルは、最後にgalKマーカーを削除します。 最後に、これらの試薬のいくつかがワームを使用していないラボで修正されたBACをまたはfosmidsの構築に使用できることがあります。 pMOD4 galK - GとpMOD4 galK - GT遺伝子は、まだその以前に発表されたオリゴはまだgalK遺伝子 15を増幅するために使用できる全体galK遺伝子を保持する。これはまだプラスミド親を消化したり、慎重にPCR産物を精製する必要がないに関してはR6Kの複製起点を使うことの利点を保持します。

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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 ...

the-production-c-elegans-transgenes-via-recombineering-with-galk

Nanyang Technological University

Research

JoVE Journal

Biology

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

14.5K ビュー.  Beth Israel Deaconess Medical Center, Harvard Medical School. のために導入遺伝子を産生する能力線虫（Caenorhabditis elegans）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。 galK選択可能なマーカー。

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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

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 ...

生物学

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

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

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

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. ...

Generation of Transgenic C. elegans by Biolistic Transformation

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 generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

トランスジェニックの世代 C.エレガンスバイオリスティック変換による

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 ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan 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 (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

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

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

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

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

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

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

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

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 including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

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

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

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

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

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

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

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

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

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