עבודה זו נתמכה על ידי קרנות מן המכונים הלאומיים לבריאות MH097163. כמה זנים סופקו על ידי CGC, אשר ממומן על ידי משרד NIH של תוכניות תשתיות מחקר (P40-OD010440).

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החוקרים מצהירים כי אין להם אינטרסים כלכליים מתחרים.

יש לנו תיארתי פרוטוקול שמשתמש בספריית האכלת dsRNA זמינה מסחרי כדי לבצע מסכי בקנה מידה גדולה בג elegans. אנו מספקים את כל ההוראות כדי לשכפל את הספרייה ולהשתמש בו ביעילות. אנו מראים כי גישה זו היא מסוגלת לזהות גני המעורבים בתהליכים ביולוגיים שונים ברקמות שונות של בעלי ה?...

מבחינה היסטורית, מסכי גנטיים בג elegans בוצעו על ידי טיפול בבעלי חיים עם mutagen כימי כגון methanesulfonate אתיל ליצור מוטציות אקראיות בתוך ה-DNA. צאצאים או grandprogeny של בעלי החיים mutagenized אז בודדו תערוכה שפנוטיפ חריג רצוי. המוטציה אחראית לגרימת פנוטיפ מכן זוהתה באמצעות תהליך מיפוי עתיר עבודה או על ידי רצף הגנום כולו של התולעת שעברה מוטציה. ישנם יתרונות רבים לביצוע מסכי mutagenesis הכימי כגון הם יוצרים נגעים קבועים בתוך הגנום התולעת שיכולה לגרום לשני פנוטיפים לזכות-of-פונקציה ואובדן של פונקציה, אבל תהליך המיפוי הוא איטי ויקר. התערבות פעמיים תקוע RNA (dsRNAi) מספקת אמצעים חלופיים כדי לזהות גני המעורבים בתהליכים ביולוגיים חשובים. בשיטה זו, dsRNA רצף הקידוד של גני אנדוגניים התאמה מוחדר לתולעת.DsRNA מעובד in vivo כדי ליצור RNAs (21-22 נוקלאוטיד) הקטן שישמש כמדריכים כדי למצוא להיקשר mRNAs אנדוגני ההתאמה, מיקוד אותם להשמדת 1. הליך זה הוא מהיר יחסית, אבל בגלל dsRNA מטרות mRNA ולא ה-DNA, רק פנוטיפים ירידה של הפונקציה הם נצפו באמצעות גישה זו. מבוא של dsRNAs לבעלי חיים יכול להיות מושגת על ידי הזרקה ישירה של dsRNA 2, על ידי שריה בחי dsRNA 3, או על ידי האכלת בעלי חיים חיידקים שמבטאים 4 dsRNA. כל אחת משיטות אספקה ​​אלה גורם לתופעות מציאה מערכתיות כמו רוב התאים של החיה להביע SID-1, ערוץ הטרנסממברני מסוגל לייבא dsRNA 5. שימש במקור כגישת גנטיקה הפוכה למציאת הביטוי של גנים בודדים אחד בכל פעם, את הפיתוח של שתי ספריות האכלה עכשיו מאפשר מסכי גנטיים בקנה מידה גדולה. DsRNA הספריות, הזמינות מסחרי מכילות baהשיבוטים cterial המייצגים אף אחד 55% או 87% מכל ג elegans גנים 6, 7. למרבה הצער, מסכי האכלת dsRNAi בקנה מידה גדולה לעתים לגרום יעילות מציאה משתנה 8-10. בעוד שהרמה המוחלטת של מציאה ידי RNAi אינה נמדדת בקלות, ביעילות מציאה מופחתת נצפתה במסכים בקנה מידה גדולים חייבת להיגרם על ידי mRNAs גן ספציפי שיורית שלברוח השפלה ידי RNAi. זה, בתורו, יכול להיות תוצאה ישירה של אובדן פלסמיד dsRNA מחיידקים נמאס לבעלי חיים במסכים אלה. תמיכה ברעיון הזה, חיות מאכילה את התערובות של חיידקים, בהם רק 50% מהחיידקים להביע dsRNAs נגד גן ממוקד, להראות מצטמצם באופן דרמטי (אם יש) השפעות מציאה כאשר לעומת השליטה האכיל חיות 11. של מציאה גן במידה הופכת לדאגה קריטית בעת ביצוע מסכי dsRNAi כמו רוב הגנים בג elegans הוא haplosufficient וכך ביטוי גנים חייב להיות מופחת על ידי t 50% לפחותo לגרום לאובדן של פונקצית פנוטיפים 12. יש לנו בשימוש בפרוטוקולי האכלה שפורסם בעבר לבצע מסכי בקנה מידה גדולה ומצאנו את אותו אפקט מציאה משתנה שדווח על ידי אחרים 8-10. בחיפוש אחר סיבות אפשריות, מצאנו כי כמעט 60% מהחיידקים האכילו את חיות במסך איבדו את הפלסמיד dsRNA. פיתחנו פרוטוקול כפילויות והקרנת ספרייה שמפחית באופן משמעותי או מבטל הפסד פלסמיד ומאוד משפר את יעילות מציאה. פרוטוקול זה מתאים לביצוע מסכי בקנה מידה גדולים בג elegans ויכול לשמש מסך אחד משתי ספריות dsRNA הזמינות המסחרית. באמצעות פרוטוקול זה, אנו מוצאים כי למעשה 100% מהחיידקים נמאס לבעלי חיים במסך לשמר את פלסמיד dsRNA הקידוד ולגרום לנשירה חזקה וpenetrant מאוד של פנוטיפים פונקציה בכל הרקמות שנבדקו.

<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

התערבות RNA על ידי האכלת חיידקי תולעים להביע dsRNAs כבר כלי שימושי כדי להעריך את תפקוד גן בג elegans. בעוד אסטרטגיה זו עובדת היטב כאשר מספר קטן של גנים ממוקדים למציאה, מסכי האכלה בקנה מידה גדולה מראים את יעילות מציאה משתנה, אשר מגבילה את השירות שלהם. יש לנו דקונסטרוקציה פרוטוקולי מציאה RNAi שפורסמו בעבר ונמצאו כי המקור העיקרי למציאה מופחתת ניתן לייחס לאובדן של פלסמידים dsRNA-קידוד מהחיידקים האכילו את בעלי החיים. בהתבסס על תצפיות אלה, פיתחנו פרוטוקול האכלת dsRNA שמקטין באופן משמעותי או מבטל הפסד פלסמיד כדי להשיג מציאה תפוקה יעילה, גבוהה. אנו מראים כי פרוטוקול זה יהיה לייצר דפיקה חזקה, לשחזור למטה של ג elegans גנים בסוגי רקמות רבים, כוללים תאי עצב, וייאפשרו מציאה יעילה במסכים בקנה מידה גדולים. פרוטוקול זה משתמש האכלת dsRNA זמינה מסחריספרייה ומתארת ​​את כל צורך לשכפל את הספרייה ולבצע מסכי dsRNA הצעדים. הפרוטוקול אינו מחייב שימוש בכל ציוד מתוחכם, ולכן יכול להתבצע על ידי כל ג elegans מעבדה.

<html> פנוטיפים מציאה P0 יתחילו להופיע אחרי 2-3 ימים של האכלת בעלי חיים dsRNA להביע חיידקים. כמה פנוטיפים מוקדם כוללים מעצר והקטלניות זחל ויש לשים לב ב2-3% מכלל בארות ההאכלה. אותם פנוטיפים יהיו גם נצפו בבעלי החיים דור F1. הנוכחות של ביצי F1 שאינן מצליחין לבקוע היא עוד פנוטיפ F1 משו?...

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

בעוד dsRNA ההאכלה בג elegans הוא כלי רב עוצמה כדי להעריך את תפקוד גן, פרוטוקולים הנוכחיים למסכי האכלה בקנה מידה גדולה יביאו ליעילות מציאה משתנה. אנו מתארים פרוטוקול משופר לביצוע מסכי האכלת RNAi בקנה מידה גדולה, כי תוצאות במציאה יעילה ולשעתקו מאוד של ביטוי גנים.

מציאה גן בקנה מידה גדולה ב<em&gt; ג elegans</em&gt; שימוש בספריות האכלת dsRNA ליצור פנוטיפים הפסד של הפונקציה חזקות

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 ...

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Research

JoVE Journal

Biology

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

11.9K Views.  University of Massachusetts, Amherst.  בעוד dsRNA ההאכלה בג elegans הוא כלי רב עוצמה כדי להעריך את תפקוד גן, פרוטוקולים הנוכחיים למסכי האכלה בקנה מידה גדולה יביאו ליעילות מציאה משתנה. אנו מתארים פרוטוקול משופר לביצוע מסכי האכלת RNAi בקנה מידה גדולה, כי תוצאות במציאה יעילה ולשעתקו מ...

Video: 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.

בקנה מידה גדול של מסכי ספריות Metagenomic

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

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.

הדור של Stable טראנסגנטי C. elegans שימוש Microinjection

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.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

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.

בקנה מידה גדול דג הזברה מבוסס ב-vivo מולקולה קטנה מסך

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 הקרנת לזהות פנוטיפים Postembryonic ב ג 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.

באמצעות רצף מעצר SecM ככלי לבודד פוליפפטידים כרוכים הריבוזום

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.

באמצעות אסטרטגית האכלת התערבות RNA בתיווך למסך לגנים מעורבים בויסות משקל גוף בנמטודות<em&gt; ג 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.

נוגדן מכתים ב<em&gt; ג Elegans</em&gt; שימוש &quot;Freeze-פיצוח&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.

שיטות להערכת subcellular תאים של שריר ב<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.

מדידת רעילות ב -C. אלגיה

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