この作品は、健康MH097163の国立研究所からの資金によって支えられている。いくつかの株は、研究基盤プログラムのNIHのオフィス（P40-OD010440）によって資金を供給されるCGC、提供された。

<ol>
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G., Ahringer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+specific+RNA-mediated+interference+through+ingested+double-stranded+RNA+in+Caenorhabditis+elegans.">Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans.</a> Genome Biol. 2 (1), (2001).</li><li>Hodgkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotype,+ploidy,+and+gene+dosage.">Karyotype, ploidy, and gene dosage.</a> WormBook. , 1-9 (2005).</li><li>Kennedy, S., Wang, D., 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=A+conserved+siRNA-degrading+RNase+negatively+regulates+RNA+interference+in+C.+elegans.">A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans.</a> Nature. 427 (6975), 645-649 (2004).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+misexpression+of+germline+P+granules+and+enhanced+RNA+interference+in+retinoblastoma+pathway+mutants.">Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants.</a> Nature. 436 (7050), 593-597 (2005).</li><li>Lackner, M. R., Nurrish, S. J., Kaplan, 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=Facilitation+of+synaptic+transmission+by+EGL-30+Gqalpha+and+EGL-8+PLCbeta:+DAG+binding+to+UNC-13+is+required+to+stimulate+acetylcholine+release.">Facilitation of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLCbeta: DAG binding to UNC-13 is required to stimulate acetylcholine release.</a> Neuron. 24 (2), 335-346 (1999).</li><li>Bastiani, C. A., Gharib, S., Simon, M. I., Sternberg, P. W. <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+Galphaq+regulates+egg-laying+behavior+via+a+PLCbeta-independent+and+serotonin-dependent+signaling+pathway+and+likely+functions+both+in+the+nervous+system+and+in+muscle.">Caenorhabditis elegans Galphaq regulates egg-laying behavior via a PLCbeta-independent and serotonin-dependent signaling pathway and likely functions both in the nervous system and in muscle.</a> Genetics. 165 (4), 1805-1822 (2003).</li><li>Lickteig, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neurotransmitter+vesicles+by+the+homeodomain+protein+UNC-4+and+its+transcriptional+corepressor+UNC-37/groucho+in+Caenorhabditis+elegans+cholinergic+motor+neurons.">Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 and its transcriptional corepressor UNC-37/groucho in Caenorhabditis elegans cholinergic motor neurons.</a> J. Neurosci. 21 (6), 2001-2014 (2001).</li><li>Terami, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+organization,+expression,+and+analysis+of+the+troponin+C+gene+pat-10+of+Caenorhabditis+elegans.">Genomic organization, expression, and analysis of the troponin C gene pat-10 of Caenorhabditis elegans.</a> J Cell Biol. 146 (1), 193-202 (1999).</li><li>Novelli, J., Page, A. P., Hodgkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C+terminus+of+collagen+SQT-3+has+complex+and+essential+functions+in+nematode+collagen+assembly.">The C terminus of collagen SQT-3 has complex and essential functions in nematode collagen assembly.</a> Genetics. 172 (4), 2253-2267 (2006).</li><li>Brundage, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+a+C.+elegans+Gqalpha+gene+disrupt+movement,+egg+laying,+and+viability.">Mutations in a C. elegans Gqalpha gene disrupt movement, egg laying, and viability.</a> Neuron. 16 (5), 999-1009 (1996).</li><li>G&#246;nczy, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+analysis+of+cell+division+in+C.+elegans+using+RNAi+of+genes+on+chromosome+III.">Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III.</a> Nature. 408 (6810), 331-336 (2000).</li><li>Miller, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=elegans+unc-4+gene+encodes+a+homeodomain+protein+that+determines+the+pattern+of+synaptic+input+to+specific+motor+neurons.">elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons.</a> Nature. 355, 841-845 (1992).</li><li>White, J. G., Southgate, E., Thomson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+Caenorhabditis+elegans+unc-4+gene+alter+the+synaptic+input+to+ventral+cord+motor+neurons.">Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons.</a> Nature. 355, 838-841 (1992).</li><li>Johnson, N. M., Behm, C. A., Trowell, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+and+inducible+gene+knockdown+in+C.+elegans+using+Wormgate+and+the+ORFeome.">Heritable and inducible gene knockdown in C. elegans using Wormgate and the ORFeome.</a> Gene. 359, 26-34 (2005).</li><li>Wani, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1+dopamine+receptor+signaling+is+modulated+by+the+R7+RGS+protein+EAT-16+and+the+R7+binding+protein+RSBP-1+in+Caenoerhabditis+elegans+motor+neurons.">D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons.</a> PLoS One. 7 (5), e37831 (2012).</li><li>Beifuss, K. K., Gumienny, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+screening+to+identify+postembryonic+phenotypes+in+C.+elegans.">RNAi screening to identify postembryonic phenotypes in C. elegans.</a> J. Vis. Exp. (60), e3442 (2012).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

我々はC.大規模スクリーニングを行うために、市販のdsRNAを送りライブラリを使用してプロトコルを記載した虫 。我々は、ライブラリを複製し、それを効果的に使用するようにすべての手順を説明します。我々は、このアプローチは、動物の異なる組織における異なる生物学的過程に関与する遺伝子を同定することができることを実証している。我々はここで説明した表現型が?...

Cの歴史的には、遺伝子スクリーニングエレガンスは、DNA内にランダム突然変異を作成するために、例えば、エチルメタンスルホネートなどの化学変異原で動物を処理することによって行われている。変異誘発された動物の子孫またはgrandprogenyは、その展示を希望する異常な表現型を分離している。表現型を引き起こす原因となる変異は、次いで、労働集約的なマッピング手順を介して、または変異型ワームの全ゲノムを配列決定することによって同定される。そこに、彼らは両方のゲイン関数のと機能喪失型の表現型を引き起こすことができるワームゲノム内の恒久的な損傷を作成するような化学的突然変異誘発画面を実行するには多くの利点がありますが、マッピング手順が遅く、高価である。 二本鎖RNA干渉（dsRNAi）の重要な生物学的過程に関与する遺伝子を同定するための代替手段を提供する。この方法では、内因性遺伝子のコード配列に一致するdsRNAはウォームに導入される。dsRNAはガイドが破壊1のためにそれらを標的化、マッチング内因性mRNAを見つけて結合するように機能する小さな（21〜22ヌクレオチド）RNAを生成するために、 生体内で処理される。この手順は、比較的迅速であるが、dsRNAがmRNAのではなく、DNAを標的とするため、唯一の還元の機能表現型は、このアプローチを使用して観察される。 動物へのdsRNAの導入は、dsRNAの3匹の動物を浸漬することにより、またはdsRNA 4を発現する動物の細菌を供給することにより、2のdsRNAの直接注射により達成することができる。これらの送達方法はそれぞれの動物のほとんどの細胞は、SID-1は、dsRNA 5のインポートが可能な膜貫通チャネルを表現するなどの全身のノックダウン効果を引き起こす。当初の時点で、個々の遺伝子の発現のいずれかをノックダウンする逆遺伝学的アプローチとして用い、2つの給電ライブラリーの開発は、現在、大規模な遺伝子スクリーニングを可能にする。市販のdsRNAはライブラリはBAが含まれているすべてのCの 55パーセント以上87パーセントのどちらかを表現cterialクローン虫の遺伝子6、7。 残念ながら、大規模なdsRNAi送り画面は、多くの場合、変数のノックダウン効率8月10日になる。 RNAiによるノックダウンの絶対レベルは容易に測定されていないが、大規模な画面で観察減少ノックダウン効率は、RNAiによる分解を免れる残留遺伝子特異的なmRNAによって引き起こされている必要があります。これは、順番に、これらの画面で動物に供給される細菌からのdsRNAのプラスミドの損失の直接の結果であってもよい。ノックダウン効果を給餌した動物11を制御するために比較した場合、（もしあれば）は、この考えを支持する、動物は、細菌の50％のみが標的遺伝子に対するdsRNAを発現する細菌の混合物を供給し、劇的に減少を示す。 Cのほとんどの遺伝子としてdsRNAi画面を実行するときに遺伝子ノックダウンの程度は重大な関心事になるエレガンス haplosufficientであり、したがって、遺伝子発現は少なくとも50％のtだけ小さくしなければならないO機能喪失は12を表現型の原因。 我々は、大規模なスクリーニングを行うために以前に公開された給餌プロトコルを使用し、他の8〜10によって報告された同一の変数ノックダウン効果を見出した。考えられる原因の検索では、画面内の動物に与えた細菌の60％近くは、dsRNAのプラスミドを失っていたことがわかった。当社は、飛躍的に低減または排除するプラスミドの損失を大幅ノックダウン効率を改善し、ライブラリの重複やスクリーニングプロトコルを開発した。このプロトコルは、C.大規模スクリーニングを実施するのに適しているエレガンスとは、市販のdsRNAライブラリーのどれかをスクリーニングするために使用することができる。このプロトコルを使用して、我々は、画面中、動物に供給された細菌の本質的に100％がdsRNAをコードするプラスミドを保持し、試験した全ての組織における機能の表現型の堅牢で高度に浸透損失を引き起こすことを発見。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>96-well Falcon flat bottom plate </td>
 <td>Fisher Scientific</td>
 <td>353072</td>
 <td>sterile</td>
 </tr>
 <tr>
 <td>adhesive foil</td>
 <td>USA Scientific</td>
 <td>2923-0100</td>
 <td></td>
 </tr>
 <tr>
 <td>Nunc omniplate</td>
 <td>Fisher Scientific</td>
 <td>267060</td>
 <td></td>
 </tr>
 <tr>
 <td>2.0 ml deep 96-well polypropylene Masterblock</td>
 <td>USA Scientific</td>
 <td>5678-0285</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>Lids for deep well plates</td>
 <td>USA Scientific</td>
 <td>5665-6101</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml combitips</td>
 <td>USA Scientific</td>
 <td>4796-5000</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>Reservoir</td>
 <td>USA Scientific</td>
 <td>2977-8500</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>12-well plates</td>
 <td>USA Scientific</td>
 <td>CC7682-7512</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>dsRNA feeding library</td>
 <td>OpenBiosystems</td>
 <td>RCE1181</td>
 <td>store -80 &deg;C</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Fisher Scientific</td>
 <td>BP1760-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetracycline</td>
 <td>Fisher Scientific</td>
 <td>BP912-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbenicillin</td>
 <td></td>
 <td></td>
 <td>allow 2-4 weeks for delivery <a href="http://www.biochemicaldirect.com" target="_blank">www.biochemicaldirect.com</a></td>
 </tr>
 <tr>
 <td>Bacteriolgical Agar Ultrapure grade A </td>
 <td>Affymetrix</td>
 <td>10906</td>
 <td>for worm plates</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Brayer roller</td>
 <td>USA Scientific</td>
 <td>9127-2940</td>
 <td></td>
 </tr>
 <tr>
 <td>Boekel 96-pin replicator</td>
 <td>Fisher Scientific</td>
 <td>05-450-9</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>microshaker</td>
 <td>Thomas Scientific</td>
 <td>1231A93</td>
 <td>3 mm orbit</td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (0.5-10 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-0510</td>
 <td></td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (30-300 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-3300</td>
 <td></td>
 </tr>
 <tr>
 <td>Repeater Plus manual pipettor</td>
 <td>USA Scientific</td>
 <td>4026-0201</td>
 <td></td>
 </tr>
 </tbody>
 </table>

large-scale gene knockdown in c. elegans using dsrna feeding libraries to generate robust loss-of-function phenotypes

dsRNAを発現するワーム細菌を供給することにより、RNA干渉は、C.での遺伝子機能を評価するための有用なツールであった虫 。少数の遺伝子がノックダウンの標的とされるときにこの戦略はうまく機能するが、大規模な給電スクリーンはそれらの有用性を制限する、可変ノックダウン効率を示している。我々は、以前に公表されたRNAiノックダウンプロトコルを解体させ、ノックダウンの主な原因は、動物に与え細菌からのdsRNAをコードするプラスミドの喪失に起因し得ることを見出した。これらの観​​察に基づいて、我々は非常に効率的な、ハイスループットノックダウンを達成するために、プラスミド損失を低減または排除するのdsRNA給餌プロトコルを開発した。我々は、このプロトコルはCのダウン、堅牢で再現性のあるノックを生成することを実証ニューロンを含む複数の組織タイプで遺伝子を、 線虫 、および大規模なスクリーニングにおいて効率的なノックダウンを可能にする。このプロトコルは、市販のdsRNA送りを使用していますライブラリとライブラリを複製したdsRNAの画面を実行するために必要なすべての手順を説明します。プロトコルは任意の高度な機器の使用を必要としないので、任意C.によって行うことができるラボエレガンス 。

P0ノックダウン表現型は、細菌を表現するdsRNA動物の飼育、2〜3日後に表示されるようになります。いくつかの初期の表現型は、幼虫の逮捕と致死性が含まれており、すべての給紙のウエル2〜3％で観察されるべきである。これらの同じ表現型はまた、F1世代の動物で観察されるであろう。孵化に失敗し、F1の卵の存在は、他の一般的なF1の表現型であり、一緒に、これらの3表現型は、F1の画面?...

Watch this Scientific Journal Video about Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes at JoVE.com

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

dsRNAは、Cで供給しながら、 エレガンスは、大規模な送り画面の現在のプロトコルは、可変ノックダウン効率をもたらす遺伝子機能を評価するための強力なツールである。我々は、遺伝子発現の非常に有効かつ再現性のノックダウンをもたらす大規模なRNAi送り画面を実行するための改良されたプロトコルを記載している。

大規模な遺伝子ノックダウン中<em&gt; C.虫</em&gt;堅牢な機能喪失表現型を生成するためのdsRNA摂食ライブラリの使用

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

large-scale-gene-knockdown-c-elegans-using-dsrna-feeding-libraries-to

Research

JoVE Journal

Biology

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

11.9K ビュー.  University of Massachusetts, Amherst.  dsRNAは、Cで供給しながら、 エレガンスは、大規模な送り画面の現在のプロトコルは、可変ノックダウン効率をもたらす遺伝子機能を評価するための強力なツールである。我々は、遺伝子発現の非常に有効かつ再現性のノックダウンをもたらす大規模なRNAi送り画面を実行するための改良されたプロトコルを記載している。 

ビデオ: Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

メタゲノムライブラリの大規模スクリーン

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

生物学

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 ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

大規模ゼブラフィッシュベース in vivoで小分子スクリーン

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

の後胚表現型を識別するためのRNAiスクリーニング C.エレガンス</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体染色中<em&gt; C.虫</em&gt;「フリーズクラッキング」使用

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

筋肉の細胞内コンパートメントを評価するための方法<em&gt; C。エレガンス</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C.エレガンスにおけるRANペプチド毒性の測定

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...