אנו מודים סמואל וורד, דיאן ג שייקים גרגורי נלסון עבור חלוצי הטכניקה הזו. אנו מודים Pavan Kadandale ובריאן Geldziler עבור קטעי וידאו המציג זרע מופעל, חברי המעבדה Singson לדיונים מועיל; CGC עבור זנים ו-NIH על תמיכתם בנו באמצעות מענק (R01HD054681).

1) העשרה של האוכלוסייה הגברית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בהתאם לצורך הניסוי, מספר גדול של זכרים ניתן להשיג על ידי העסקת אחת האסטרטגיות הבאות: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> אוכלוסייה גדולה של גברים סוג בר ניתן להשיג על ידי המעבר 5 זכרים סוג בר 1 אנדרוגינוס על הדשא קטן של OP50 seeded במרכז צלחת NGM. בערך 50% של בני הדור שלאחר יהיה סוג הזכרים בטבע. </li><li style=";text-align:right;direction:rtl"> אותו-5 (e1490) או אותו-8 (e1489) הרמפרודיטים לזרוק מספר רב של גברים. אותו-5 ו-8 לו זכרים פוריים ואין פגם ברור במורפולוגיה הזרע ותפקודו. לכן, אותו-5-8 או אותו הזכרים יכולים לשמש במקום הזכרים סוג בר ניסויים רבים. </li></ol></li></ol> 2) זיהוי ובידוד של זכרים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בדוק את המורפולוגיה הזנב של תולעים, תולעת הזנב של הזכר הוא מעוגל. </li><li style=";text-align:right;direction:rtl"> פיק L4 הזכרים הבמה ולהעבירם seeded צלחת NGM עם E. coli OP50 ולתת להם לגדול במשך יום או יומיים. הזכרים מתנזר גידול בהעדר הרמפרודיטים למנוע אובדן של זרע ולכן מספר גדול של spermatids יהיה זמין במהלך הליך ניסיוני. </li><li style=";text-align:right;direction:rtl"> ביום לנתיחה, להעביר את הזכרים מתנזר על צלחת NGM ללא חיידקים ולתת להם לזחול בשביל כמה דקות. פעולה זו מסייעת להסיר שכבה קטנה של E. החיידק דבק על פני השטח של תולעים. לחלופין, נפח פיפטה קטנה של M9 חיץ (6 גרם Na 2 HPO 4, 3G KH 2 PO 4, 5 גרם NaCl, 0.25g MgSO 4 0.7 H 2 O לליטר) בכוס לצפות הזכרים להעביר לתוך המאגר. </li></ol> 3) Dissection של הזכרים והן הפעלה חוץ גופית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קח שקופיות הזכוכית לסמן עיגול קטן באמצעות עט פאפ. פיפטה 30-50 μl של זרע בינוני 1X (HEPES 50mm, 25mm KCl, NaCl 45mm, 1mm MgSO 4, 5 מ&quot;מ CaCl 2, 10mm דקסטרוז, pH 7.8) בתוך מעגל. המעגל הידרופובי עוזר לשמר את הזרע בינוני בתוך הגבול שלה. </li><li style=";text-align:right;direction:rtl"> העברת הזכרים 50-10 על המדיום הזרע. </li><li style=";text-align:right;direction:rtl"> הצגת שקופיות תחת מיקרוסקופ, להחזיק שני מזרקים מחוברים עם 27 מחטים מד על שתי הידיים. </li><li style=";text-align:right;direction:rtl"> השתמש באחת המחט &quot;להחזיק&quot; את התולעת ולהשתמש מחט אחרים לחתוך את הקצה האחורי של הזכרים לשחרר spermatids. </li><li style=";text-align:right;direction:rtl"> חיתוך התולעת בדרך כלל מגרש מספר עצום של spermatids מיידי. Spermatids נראים כמו דקה &quot;גרגרי&quot; תחת מיקרוסקופ הסטריאו. עם זאת, לפעמים חלק או רוב spermatids עשוי להישמר בתוך הפגר. במקרה כזה, בעדינות &quot;גרירת&quot; פגר על שקופיות הזכוכית עשויה להקל על שחרורו של spermatids מן השלד. </li><li style=";text-align:right;direction:rtl"> כדי להפעיל את spermatids, לנתח את התולעים בינוני זרע המכיל E 1X Pronase (20μg/ml) ולתת להם לשבת במשך 5 דקות. </li><li style=";text-align:right;direction:rtl"> אל תאפשר השקופיות להיות יבש בכל נקודת זמן הניסוי. הוסף בינוני זרע 1X יותר במידת הצורך. </li></ol> 4) ויזואליזציה של spermtids ו זרע <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעדינות למקום coverslip על פני השטח של מדגם גזור. </li><li style=";text-align:right;direction:rtl"> מצא את תחום spermatids בהגדלה נמוכה (10X) </li><li style=";text-align:right;direction:rtl"> הפרטים הקטנים של spermatids או זרע ניתן לראות על ידי מעבר ההגדלה גבוה יותר - בדרך כלל טבילה שמן 100x. </li></ol> 5) נציג תוצאות דוגמה תמונה של DIC spermatids מבודד זכר C. elegans מוצג באיור 1. Spermatids הם כדוריים בצורת גרעינים בולטים. הופעל בשנת זרע במבחנה מוצגות באיור 2. <img src="/files/ftp_upload/2336/2336fig1.jpg" alt="איור 1" /> באיור 1. DIC תמונה של ג elegans spermatids. <img src="/files/ftp_upload/2336/2336fig2.jpg" alt="איור 2" /> איור 2. DIC תמונה של במבחנה מופעל C. elegans זרע.

<ol>
	<li>Kimble, J., Crittenden, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+of+germline+stem+cells,+entry+into+meiosis,+and+the+Sperm/Oocyte+decision+in+Caenorhabditis+elegans.">Controls of germline stem cells, entry into meiosis, and the Sperm/Oocyte decision in Caenorhabditis elegans.</a> Annual Review of Cell and Developmental Biology. 23, 405-433 (2007).</li><li>Lhernault, S. W., Roberts, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. , 273-301 (1995).</li><li>Hodgkin, J., Horvitz, H., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondisjunction+Mutants+of+the+Nematode+Caenorhabditis+elegans.">Nondisjunction Mutants of the Nematode Caenorhabditis elegans.</a> Genetics. 91, 67-94 (1979).</li><li>Altun, Z. F., Hall, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+male+C.+elegans+anatomy.">Introduction to male C. elegans anatomy.</a> WormAtlas. , (2008).</li><li>Shakes, D., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+spermiogenesis+in+C.+elegans:+a+pharmacological+and+genetic+analysis.">Initiation of spermiogenesis in C. elegans: a pharmacological and genetic analysis.</a> Dev Biol. 134, 189-200 (1989).</li><li>Nelson, G. A., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+fusion,+pseudopod+extension+and+amoeboid+motility+are+induced+in+nematode+spermatids+by+the+ionophore+monensin.">Vesicle fusion, pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin.</a> Cell. 19, 457-464 (1980).</li><li>L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+cell+biology+of+spermatogenesis+in+the+nematode+C.+elegans.">The genetics and cell biology of spermatogenesis in the nematode C. elegans.</a> Molecular and Cellular Endocrinology. 306, 59-65 (2009).</li><li>Chatterjee, I., Richmond, A., Putiri, E., Shakes, D., Singson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Caenorhabditis+elegans+spe-38+gene+encodes+a+novel+four-pass+integral+membrane+protein+required+for+sperm+function+at+fertilization.">The Caenorhabditis elegans spe-38 gene encodes a novel four-pass integral membrane protein required for sperm function at fertilization.</a> Development. 132, 2795-2808 (2005).</li><li>Xu, X., Sternberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+C.+elegans+sperm+TRP+protein+required+for+sperm-egg+interactions+during+fertilization.">A C. elegans sperm TRP protein required for sperm-egg interactions during fertilization.</a> Cell. 114, 285-297 (2003).</li><li>Nelson, G., Roberts, T., Ward, S. <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+spermatozoan+locomotion:+amoeboid+movement+with+almost+no+actin.">Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin.</a> J Cell Biol. 92, 121-131 (1982).</li><li>Machaca, K., DeFelice, L. J., L'Hernault, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+chloride+channel+localizes+to+Caenorhabditis+elegans+spermatids+and+chloride+channel+blockers+induce+spermatid+differentiation.">A novel chloride channel localizes to Caenorhabditis elegans spermatids and chloride channel blockers induce spermatid differentiation.</a> Dev Biol. 176, 1-16 (1996).</li><li>LaMunyon, C., Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+viability+of+mutant+and+manipulated+sperm+by+artificial+insemination+of+Caenorhabditis+elegans.">Assessing the viability of mutant and manipulated sperm by artificial insemination of Caenorhabditis elegans.</a> Genetics. 138, 689-692 (1994).</li></ol>

אין ניגודי אינטרסים הכריז.

בנוסף לניתוח מוטנטים SPE, פרוטוקול זה יש יישומים חשובים אחרים, כגון ניתוח מורפולוגיה הזרע עם ההזדקנות. Spermatids ו זרע בודד בפרוטוקול זה יכול לשמש בניסויים במורד אחרים כגון, immunostaining מסוג בר זרע מוטנטי 8, 9, תנועתיות הזרע על 10 שקופיות, מדידות פיזיולוגיות 9, 11,...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Tuberculin syringe</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>309623</td>
<td>Syringe: 1 ml, needle: 27G X &frac12; in</td>
</tr>
<tr>
<td>Pap pen</td>
<td>&nbsp;</td>
<td>Zymed</td>
<td>00-8888</td>
<td>&nbsp;</td>
<tr>
<td>VWR VistaVision HistoBond Microscope Slides</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>16004-406</td>
<td>Dimension: 75X25X1mm</td>
</tr>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>48366045</td>
<td>&nbsp;</td>
<tr>
<td>Protease</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-6911</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation and in vitro activation of caenorhabditis elegans sperm

זכרים הרמפרודיטים הם שתי צורות המינית נמצא באופן טבעי נמטודות C. elegans. זרע amoeboid מיוצרים על ידי שני גברים הרמפרודיטים. בשלב מוקדם יותר של gametogenesis, בתאי הנבט של הרמפרודיטים להתמיין מספר מוגבל של הזרע - בערך 300 - ו מאוחסנים &quot;תיק&quot; קטן שנקרא spermatheca. מאוחר יותר, הרמפרודיטים הרף לייצר ביציות 1. לעומת זאת, הזכרים מייצרים זרע רק לאורך הבגרות שלהם. הזכרים מייצרים זרע כל כך הרבה חשבונות עבור 50%&gt; של תאים הכולל 2 מבוגרים אופיינית תולעת. לכן, בידוד זרע מהזכרים קל יותר מזה של הרמפרודיטים. רק אחוז קטן של גברים נוצרות באופן טבעי, בשל הפער הלא ספונטני של כרומוזום X 3. מעבר הרמפרודיטים עם זכרים או בצורה נוחה יותר, את ההקדמה של מוטציות כדי להצמיח אותו (שכיחות גבוהה של זכרים) פנוטיפ כמה אסטרטגיות דרכו ניתן להעשיר את אוכלוסיית הגברים 3. זכרים ניתן להבחין בקלות על ידי התבוננות 4 מורפולוגיה הזנב הרמפרודיטים. הזנב של הרמפרודיט הוא הצביע, ואילו זנבו של הזכר הוא מעוגל עם מבנים ההזדווגות. חיתוך הזנב משחרר מספר עצום של spermatids מאוחסן בתוך מערכת הרבייה של הגבר. Dissection מבוצע תחת מיקרוסקופ סטריאופוני באמצעות מחטים מד 27. מאז spermatids אינם מחוברים פיזית עם כל שאר התאים, הלחץ ההידראולי מגרשת תוכן פנימי של הגוף הגברי, כולל spermatids 2. הזכרים גזור ישירות על ירידה קטנה של &quot;בינוני זרע&quot;. Spermatids רגישים שינוי ב-pH. לפיכך, HEPES, מתחם עם קיבולת חציצה טוב משמש בתקשורת הזרע. גלוקוז ומלחים אחרים מציגים בתקשורת זרע לסייע בשמירה על הלחץ האוסמוטי כדי לשמור על שלמות של הזרע. פוסט meiotic בידול של spermatids לתוך זרע נקרא spermiogenesis או הפעלה הזרע. מנענע 5, ונלסון 6 בעבר הראו כי spermatids עגול יכול להיגרם להתמיין זרע על ידי הוספת מרכיבים שונים, כולל הפעלת Pronase E. כאן אנו מדגימים במבחנה spermiogenesis של C. elegans spermatids באמצעות Pronase E. Spermiogenesis מוצלח הוא מראש הנדרשים לצורך פוריות ומכאן מוטנטים פגום spermiogenesis הם עקרים. מספר מוטציות עד כה הוכחו פגום במיוחד בתהליך spermiogenesis 7. חריגות שנמצאו במהלך בהפעלת במבחנה של הרומן SPE (spermatogenesis פגום) מוטנטים יעזור לנו לגלות שחקנים נוספים שהשתתפו באירוע.

Watch this Scientific Journal Video about Isolation and In vitro Activation of Caenorhabditis elegans Sperm at JoVE.com

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

פרוטוקול עבור בידוד והפעלת spermatids מן הזכר<em&gt; ג elegans</em&gt; מתוארת כאן. חיתוך הקצה האחורי של spermatids משחרר זכר. Spermatids יכול להיות מופעל על ידי תוספת של פרוטאז.

בידוד במבחנה הפעלת Caenorhabditis elegans זרע

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

isolation-and-in-vitro-activation-of-caenorhabditis-elegans-sperm

Research

JoVE Journal

Biology

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

16.2K Views.  Rutgers University. פרוטוקול עבור בידוד והפעלת spermatids מן הזכר ג elegans מתוארת כאן. חיתוך הקצה האחורי של spermatids משחרר זכר. Spermatids יכול להיות מופעל על ידי תוספת של פרוטאז.

Video: Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

Solid פלייט מבוססי הגבלת תזונתיים ב Caenorhabditis elegans</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

זמן לשגות מיקרוסקופית של העובר בשלב מוקדם Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

בידוד ערפדים של נוזלים מתאיים<em&gt; Elegans Caenorhabditis</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

בסיסי<em&gt; Caenorhabditis elegans</em&gt; שיטות: סנכרון תצפית

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

הפקה וניתוח בפרוסטגלנדין<em&gt; Elegans Caenorhabditis</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

טיפוח<em&gt; Elegans Caenorhabditis</em&gt; ב בשלושה ממדים במעבדה

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

משולב נוקלאוטיד שיחולץ חלבון ב Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

מדידה של קצב הצריכה של חמצן בשלם Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

טיהור וניתוח מסחטות של האלקאים ושלפוחיות

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

דוגמנות גיל הקשורים מחלות ניווניות בתוך האלאים

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...