פרוטוקול זה פותח במקור על ידי Xiaolan יה מיכאל Petrascheck על פרד האצ&#39;ינסון לחקר הסרטן בסיאטל במעבדה של לינדה באק. הגרסה המפורטת לעיל הוכנה על מנת להפוך את התהליך כולו לרשות הציבור הרחב. אנו מודים קרול טיילור, שרה LeBoeuf ואנדי Tomacelli עבור אנושות בקריאת פרוטוקול. זהו כתב היד # 2866 מן המחקר סקריפס המכון. המעבדה Petrascheck ממומנת על ידי תוכנית נוברטיס ADI.

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אין ניגודי אינטרסים הכריז.

פרוטוקול שהוצג מאפשר מדידת C.elegans תוחלת החיים ב 96 צלחות microtiter היטב. כפי שמוצג בתוצאות נציג בסעיף זה אמין משכפל ממצאים קודמים ומספק מידע כמותי. שימוש assay זה יש לנו הוקרן בהצלחה והשפעתם על תוחלת החיים C.elegans מעל 89,000 מולקולות. לצורך ההקרנה סמים, מדידת תוחלת החיים בתבנית 96 microtiter גם צלחת יש מספר יתרונות על פני assay התקשורת הקלאסית מוצק. זה מפחית את העבודה הדרושה התקשורת הכנה, את כמות שטח הדגירה, ואת כמות התרופה הנדרשת. הפורמט 96-היטב את ההגדרה מיקרוסקופיה לאפשר אוטומציה של assay כולו הקרנות תפוקה גבוהה. במהלך הפיתוח של assay ערכים מרובים עבור כל משתנה ושילובים של הנ&quot;ל נבדקו השפעתם על C.elegans תוחלת החיים. בדיקות אלה כללו ריכוז OP50 שונים הנעים בין 3-10 מ&quot;ג / מ&quot;ל ​​(3, 4, 6, 8, 10 מ&quot;ג / מ&quot;ל), מספר שונה של תולעים בכל טוב החל 7-45 (7, 10, 15, 22, 45 תולעים / טוב), כרכים תרבות שונים הנעים בין 40 ל 150 לכל μL היטב (40, 60, 80, 100, 120, 150 μL), וכן שינויים בהרכב חיץ. אם נמאס עם OP50 Carbenicillin עמידים, ציין לא Carbenicillin ולא amphotericin B בריכוזים נמצאו להשפיע C.elegans תוחלת החיים. רעד עדין רציף של הצלחות, כמו מומלץ לעיתים קרובות בתרבות C.elegans נוזלי, נמצא לוותר על בנפחים קטנים המשמשים assay זה. רעד עדין אך נדרש אם microtiter צלחות עם בארות גדולים יותר לשמש. הערכים המצוינים בפרוטוקול זה נבחרו בקפידה על בסיס השוואה סטטיסטית של תנאים שונים שנבדקו. התכונה המפתיעה ביותר של assay זה כנראה העובדה C.elegans יכולים להישמר 96 צלחות היטב חתומות עם הקלטת. Side-by-הצד השוואות לא גילה כל הבדל בין תוחלת החיים של חיות בתרבית unsealed צלחות, צלחות חתום עם אוטם ניילון, או צלחות חתום עם חומרי איטום המאפשרים חילופי אוויר. בכל שלושת התנאים, החיות פיתחו homogenously מאוד L1 למבוגרים הרה להולדת בתוך 65 שעות והראה תוחלת החיים דומה מאוד. בעלי חיים גדל במקביל על NGM פיתחו מעט מהר יותר, אבל ההבדל הזה היה עצמאי של היעדרות או נוכחות של אוטם. הפרוטוקול שהוצג מבוסס על חיידקים חיים, אבל יכול להיות מותאמים עבור חיידקים מתים. עם זאת, assay נוזל חיידקים לשרוד אחד יכול להתרבות במהירות לאכלס מחדש את התרבות. בידיים שלנו, הדרך היחידה אמין כדי להרוג חיידקים עד כדי כך שהם יכולים לשמש לתרבות נוזלי על ידי טיפול ממושך של החיידקים עם קרינת גמא. נקודה אחת חשוב לקחת בחשבון בתכנון של ניסוי תוחלת החיים הוא הכוח של גילוי. תלוי בגודל ההשפעה, ההשפעה של התרופה עניין חייבים להיבדק בבארות מרובים. ב assay טיפוסי 4 סמים מטופלים 4 בארות שליטה, המקביל בערך 40-50 חיות כל, צריך להספיק כדי לזהות עלייה של 30% בתוחלת החיים ב 95% יותר מאשר הניסויים. עליות של 14% מזוהים רק 60% מהמקרים ולכן דורשים בארות לשכפל יותר. שפע של נתוני תוחלת החיים שנוצר על ידי assay זה עשוי לשמש לפיתוח הניסוי או זן ספציפי מודלים Gompertz מצדי 1. אלה מודלים Gompertz מועילים כדי לקבוע את הכוח של איתור כדי להעריך את מספר תוצאות חיוביות וגם שליליות שווא עבור בקנה מידה גדול המסכים. אנו לאמת את התחזיות של מודלים אלה בניסויים עיוור והשתמשו בהם כדי להעריך את מספרם של תשלילים כוזב מסכים גדולים (תוצאות לא פורסם). לסיכום, אנו צופים כי assay הציג תהיה תועלת רבה לזהות מולקולות קטנות להאריך את תוחלת החיים של C.elegans ו ללמוד את המנגנונים.

A correction to the article Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates was made.

Erratum: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Amphotericin B</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>cat# 171375</td>
<td>Also called Fungizone</td>
</tr>
<tr>
<td>FUDR</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>cat# F0503</td>
<td>FUDR is inactivated by heat, thaw in cold water</td>
</tr>
<tr>
<td>Mianserin</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>M2525-250MG</td>
<td>Use as positive control at 50&mu;M final concentration</td>
</tr>
<tr>
<td>Cyproheptadine</td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>cat#C6022-MG</td>
<td>Alternative positive control at 10 &mu;M final concentration</td>
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<td>Sealing Tape</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>cat# 236370</td>
<td>Polyester, non-sterile</td>
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<tr>
<td>96 well plate</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>cat# 351172</td>
<td>Non-treated, transparent, sterile individual packaged, polystyrene</td>
</tr>
<tr>
<td>Nunclon flask</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>cat# 178883</td>
<td>used for large volumes</td>
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measuring caenorhabditis elegans life span in 96 well microtiter plates

תוחלת החיים היא תהליך ביולוגי מוסדר על ידי מספר מסלולים גנטיים. אסטרטגיה אחת כדי לחקור את הביולוגיה של ההזדקנות היא ללמוד חיות הנמל מוטציות מרכיבי גיל הרגולציה המסלולים. אם מוטציות אלו לטרוד את הפונקציה של המסלול לגיל הרגולציה, ולכן לשנות את תוחלת החיים של האורגניזם כולו, הם מספקים תובנות מכניסטית חשוב 1-3. אסטרטגיה נוספת לחקור את הרגולציה של תוחלת החיים היא להשתמש מולקולות קטנות להפריע גיל הרגולציה המסלולים. נכון להיום, מספר מולקולות ידועים להאריך את תוחלת החיים ביצורים שונים מודל משמשים ככלים ללמוד את הביולוגיה של ההזדקנות 4-16. מספר מולקולות מזוהה עד כה הוא קטן לעומת &quot;הכלים&quot; הגנטי הזמין מחקר על הביולוגיה של ההזדקנות. Caenorhabditis elegans היא אחד הדגמים העיקרון המשמש במחקר ההזדקנות בגלל גנטיקה מעולה שלה אורך חיים קצר של שלושה שבועות. לאחרונה, C.elegans התפתחה כאורגניזם מודל מבוסס פנוטיפ מסכי סמים 5,7,16-20 בגלל גודלו הקטן ואת יכולתה לצמוח הצלחות microtiter. כאן אנו מציגים assay למדוד C.elegans תוחלת החיים ב 96 צלחות microtiter היטב. Assay פותחה בהצלחה למסך ספריות גדולות של מולקולות להאריך את תוחלת החיים C.elegans 7. האמינות של assay הוערך בדיקות מרובות: ראשית, על ידי מדידת תוחלת החיים של חיות הבר סוג גדל בטמפרטורות שונות; השני, על ידי מדידת תוחלת החיים של מוטנטים עם תוחלת החיים משתנה; שלישי, על ידי מדידת השינויים תוחלת החיים בתגובה ריכוזים שונים Mirtazepine של דיכאון. Mirtazepine יש בעבר הוכח להאריך את תוחלת החיים ב C.elegans 7. התוצאות של בדיקות אלה מראים כי assay הוא מסוגל לשכפל את הממצאים הקודמים של מבחני אחרות הוא כמותי. הפורמט microtiter גם עושה את זה assay תוחלת החיים תואם למערכות אוטומטיות טיפול נוזלי מאפשר השתלבות בפלטפורמות אוטומטיות.

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Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

בפרוטוקול זה אנו מציגים שיטה למדידת<em&gt; Caenorhabditis elegans</em&gt; תוחלת החיים בשנת 96 צלחות microtiter היטב.

מדידת Caenorhabditis elegans חיים טווח של 96 צלחות microtiter טוב

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

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Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

36.0K Views.  The Scripps Research Institute. בפרוטוקול זה אנו מציגים שיטה למדידת Caenorhabditis elegans תוחלת החיים בשנת 96 צלחות microtiter היטב.

Video: Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

מדידת Caenorhabditis elegans חיים טווח על מדיה מוצק

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

כימות כרונולוגי שמרים תוחלת החיים על ידי תולדה של תאים בני

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

מדידת תוחלת החיים בשמרים replicative הנצה

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

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 ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

Ice יתר: שיטה גידול ארבידופסיס צמחים עגבניות ב 96-פלטות היטב genotyping תפוקה גבוהה

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

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 ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

מדידת התנגדות סטרס חמצוני של<em&gt; Elegans Caenorhabditis</em&gt; בצלחות microtiter 96-היטב

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

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 ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

שימוש באלגנים של Caenorhabditis למסך עבור אינטראקציות ממלווים ספציפיות לרקמות

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...