Diese Arbeit wurde durch Mittel aus den National Institutes of Health MH097163 unterstützt. Einige Stämme wurden von der CGC, die von der NIH Office of Research Infrastructure Programme (P40-OD010440) gefördert wird.

<ol>
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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. 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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. 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Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Wir haben ein Protokoll, das einen im Handel erhältlichen Futter dsRNA-Bibliothek verwendet, um große Bildschirme in C durchzuführen beschrieben elegans. Wir bieten alle Anweisungen, die Bibliothek zu kopieren und es effektiv zu nutzen. Wir zeigen, dass dieser Ansatz in der Lage ist, Gene in verschiedenen biologischen Prozessen in verschiedenen Geweben der Tiere beteiligt sind. Während die Phänotypen wir hier beschreiben, sind leicht zu beobachten (UNC, Egl und Dpy), kann dieses Protokoll angepass...

Historisch genetische Screens in C. elegans durch die Behandlung von Tieren mit einem chemischen Mutagen wie Ethylmethansulfonat um zufällige Mutationen in DNA erstellen durchgeführt worden. Nachkommen-oder grandprogeny mutagenisierter Tiere werden dann isoliert, die Ausstellung eines gewünschten abnorme Phänotyp. Der zum Bewirken des Phänotyps verantwortlich Mutation in einem arbeitsintensiven Abbildungsverfahren oder durch Sequenzierung der mutierten Wurm gesamte Genom identifiziert. Es gibt viele Vorteile bei der Durchführung dieser chemischen Mutagenese-Läsionen, wie sie permanent innerhalb des Genoms, die Schnecke in beiden gain-of-function-und Verlust-von-Funktion Phänotypen erzeugen kann, aber die Zuordnungsverfahren ist langsam und teuer. Doppelsträngige RNA-Interferenz (dsRNAi) stellt ein alternatives Mittel, um Gene in wichtigen biologischen Prozessen beteiligt sind. Bei diesem Verfahren wird entsprechend der dsRNA kodierende Sequenz eines endogenen Gens in das Schnecken eingeführt.Die dsRNA wird in vivo verarbeitet, um kleine (21-22 Nukleotid) RNAs, die als Führer zu finden und zu binden, die passenden endogenen mRNAs, anzusprechen, für die Zerstörung 1 dienen generieren. Dieses Verfahren ist relativ schnell, sondern weil dsRNA zielt mRNA statt DNA werden nur Reduktions-of-function-Phänotypen mit Hilfe dieses Ansatzes beobachtet. Einführung von dsRNA in ein Tier durch direkte Injektion von dsRNA 2 erreicht werden, durch Einweichen in Tieren dsRNA 3, oder durch Füttern von Tieren Bakterien, 4 dsRNA exprimieren. Jede dieser Versandmethoden verursachen systemische Zuschlags Auswirkungen, da die meisten Zellen des Tieres äußern SID-1, eine Transmembrankanal importiert werden können dsRNA 5. Ursprünglich als Reverse Genetik Ansatz, um die Expression einzelner Gene ein zu einer Zeit Knockdown verwendet, die Entwicklung der beiden Speise Bibliotheken ermöglicht nun groß angelegte genetische Bildschirme. Die im Handel erhältlichen dsRNA Bibliotheken enthalten bacterial Klone, die entweder 55% oder 87% aller C. elegans Gene 6, 7. Leider Groß dsRNAi Fütterung Bildschirme oft in variablen Zuschlagswirkungsgrade 8-10 führen. Während die absolute Höhe der Zuschlags durch RNAi ist nicht leicht zu messen, muss die in großen Bildschirmen beobachtet reduziert Zuschlags Effizienz durch Rest Gen-spezifischen mRNAs, die Verschlechterung zu entkommen durch RNAi verursacht werden. Dies könnte wiederum ein direktes Ergebnis der dsRNA Plasmid Verlust von Bakterien, Tieren in diesen Bildern zugeführt werden. Die Unterstützung dieser Idee, Tiere verfüttert Mischungen von Bakterien, in der nur 50% der Bakterien auszudrücken dsRNA gegen eine Zielgens, zeigen drastisch reduziert (wenn überhaupt) Knockdown-Effekte im Vergleich zu Tieren gefüttert 11 zu steuern. Das Ausmaß der Gen-Knockdown zu einem kritischen Anliegen bei der Durchführung dsRNAi-Bildschirme wie die meisten Gene in C. elegans sind haplosufficient und somit muss der Genexpression durch mindestens 50% t reduziert werden,o führen loss-of-function Phänotypen 12. Wir haben bisher veröffentlichten Fütterungsprotokolle verwendet werden, um große Bildschirme durchführen und fand die gleiche Variable Zuschlags Effekte von anderen 10.08 ausgewiesen. Bei der Suche nach möglichen Ursachen, fanden wir, dass fast 60% der Tiere in den Bildschirm eingespeist Bakterien hatten die dsRNA-Plasmid verloren. Wir haben eine Bibliothek Vervielfältigung und Screening-Protokoll, die drastisch reduziert oder eliminiert Plasmid Verlust und verbessert die Zuschlags Effizienz entwickelt. Dieses Protokoll ist für die Durchführung von Großbildschirmen in C. elegans und können zum Screening einer von der kommerziell erhältlichen dsRNA Bibliotheken werden. Über dieses Protokoll, finden wir, dass praktisch 100% der zu den Tieren während der Bildschirm eingespeist Bakterien behalten die dsRNA-kodierenden Plasmid verursachen und robust und sehr penetrant Verlust der Funktion Phänotypen in allen untersuchten Geweben.

<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

RNA-Interferenz, indem Würmer Bakterien, die dsRNA ist ein nützliches Werkzeug, um Genfunktionen in C. bewerten elegans. Während diese Strategie funktioniert gut, wenn eine kleine Anzahl von Genen für die Zuschlags gezielte, Groß Fütterung Bildschirme zeigen variable Zuschlagswirkungsgrade, was ihre Verwendbarkeit einschränkt. Wir haben zuvor veröffentlichten Protokollen RNAi Zuschlags zerlegt und festgestellt, dass die Hauptquelle der reduzierten Zuschlags kann zum Verlust von dsRNA kodierende Plasmide aus den an die Tiere verfüttert werden Bakterien zugeschrieben werden. Basierend auf diesen Beobachtungen haben wir eine dsRNA Fütterungsprotokoll, das stark reduziert oder eliminiert Plasmid Verlust effiziente Hochdurchzuschlags erreichen entwickelt. Wir zeigen, dass dieses Protokoll wird robust, reproduzierbar Knock Down von C. produzieren elegans Gene in mehreren Gewebetypen, einschließlich Neuronen und eine effiziente Knockdown in Großbildschirme ermöglichen. Dieses Protokoll verwendet eine kommerziell erhältliche dsRNA ZufuhrBibliothek und beschreibt alle notwendigen Schritte, um die Bibliothek zu kopieren und auszuführen dsRNA-Bildschirme. Das Protokoll erfordert nicht die Verwendung von hoch entwickelten Geräten, und kann daher von einem C durchgeführt werden elegans Labor.

P0 Zuschlags Phänotypen beginnt nach 2-3 Tagen der Fütterung der Tiere dsRNA exprimierenden Bakterien erscheinen. Einige frühe Larven Phänotypen gehören Verhaftung und Letalität und sollte in 2-3% aller Brunnen Fütterung beobachtet werden. Diese gleichen Phänotypen wird auch in der F1-Generation Tieren beobachtet werden. Die Anwesenheit von F1 Eier, die nicht zu schlüpfen ist eine weitere gemeinsame F1 Phänotyp, und gemeinsam werden diese drei Phänotypen in fast 10% aller Brunnen in F1 Bildschirmen beobachtet...

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

Während dsRNA Fütterung in C. elegans ist ein leistungsfähiges Werkzeug, um Genfunktionen, aktuellen Protokolle für große Bildschirme Fütterung führen zu variablen Zuschlags Effizienz zu beurteilen. Wir beschreiben eine verbesserte Protokoll für die Durchführung groß angelegte RNAi Fütterung Bildschirme, die in hochwirksame und reproduzierbare Zuschlags der Genexpression resultiert.

Groß Gene Knockdown in<em&gt; C. elegans</em&gt; Verwenden dsRNA Fütterung Bibliotheken zu Robust Loss-of-function Phänotypen generieren

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 Aufrufe.  University of Massachusetts, Amherst.  Während dsRNA Fütterung in C. elegans ist ein leistungsfähiges Werkzeug, um Genfunktionen, aktuellen Protokolle für große Bildschirme Fütterung führen zu variablen Zuschlags Effizienz zu beurteilen. Wir beschreiben eine verbesserte Protokoll für die Durchführung groß angelegte RNAi Fütterung Bildschirme, die in hochwirksame und reproduzierbare Zuschlags der Genexpression resultiert. 

Serie: 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.

Large-Scale-Bildschirme von Metagenombanken

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

Forschung

Biologie

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.

Generierung von stabilen transgenen C. elegans mit Mikroinjektion

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.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

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.

Large Scale Zebrafisch-Based In-vivo- Small Molecule-Screen

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.

RNAi-Screening auf postembryonalen Phänotypen in Identifizieren C elegans</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.

Mit SECM Arrest Sequenz als Werkzeug zum Ribosom gebunden Polypeptide isolieren

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.

Mit RNA-Interferenz Feeding Strategy to Screen for Genes in Body Größe der Rechtsetzung in der Nematode Beteiligte<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 ...

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.

Antikörperfärbung in<em&gt; C. Elegans</em&gt; Mit der &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 ...

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.

Methoden zur subzellulärer Kompartimente der Muskel in Beurteilen<em&gt; C. elegans</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.

Messung der RAN-Peptidtoxizität in C. elegans

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