وضعت أصلا من هذا البروتوكول في مركز فريد هاتشينسون لأبحاث السرطان في سياتل ويي Xiaolan Petrascheck مايكل في مختبر ليندا باك. وقد أعد هذا الإصدار المفصلة أعلاه إلى اتخاذ جميع الإجراءات المتاحة للالمجتمع الأوسع. نشكر كارول تايلور ، LeBoeuf سارة وTomacelli اندي لقراءة نقدية للبروتوكول. هذا المخطوط # 2866 من معهد سكريبس للأبحاث. ويتم تمويل مختبر Petrascheck بواسطة برنامج ADI نوفارتيس.

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الإعلان عن أي تضارب في المصالح.

قدمت بروتوكول يسمح بقياس عمر C.elegans في 96 لوحات microtiter جيدا. كما هو مبين في نتائج ممثل القسم أن يعيد موثوق النتائج السابقة ، ويوفر معلومات كمية. باستخدام هذا الاختبار وفحص أكثر من 89000 نجحنا جزيئات من حيث تأثيرها على عمر C.elegans. لغرض فحص المخدرات ، وقياس عمر في 96 لوحة تنسيق جيد microtiter ديها العديد من المزايا خلال فحص وسائل الاعلام الصلبة الكلاسيكية. أنه يقلل من العمالة المطلوبة لإعداد وسائل الإعلام ، مقدار المساحة الحضانة ، وكمية المخدرات المطلوبة. شكل 96 - جيدا والإعداد المجهري تسمح أتمتة فحص كامل للعروض إنتاجية عالية. خلال تطوير فحص قيم متعددة لكل متغير وخليط منها واختبارها لآثارها على عمر C.elegans. وشملت هذه الاختبارات OP50 تركيز مختلفة تتراوح من 3 إلى 10 ملغ / مل (3 ، 4 ، 6 ، 8 ، 10 ملغ / مل) ، عدد من الديدان المختلفة لكل بئر تتراوح بين 7 إلى 45 (7 ، 10 ، 15 ، 22 ، 45 الديدان / جيد) ، وأحجام مختلفة تتراوح الثقافة 40-150 ميكرولتر لكل بئر (40 ، 60 ، 80 ، 100 ، 120 ، 150 ميكرولتر) ، والتغيرات في تركيبة العازلة. إذا تم العثور على تغذية مع OP50 كربنيسيلين المقاوم ، لا كربنيسيلين ولا باء الامفوتريسين في تركيزات اشارت الى تؤثر C.elegans العمر. تم العثور على الهز المستمر لطيف من لوحات ، على النحو الموصى به في كثير من الأحيان في الثقافة C.elegans السائل ، والاستغناء عن وحدات التخزين الصغيرة المستخدمة في هذا الاختبار. تهتز لطيف ولكن إذا كان هناك حاجة لوحات microtiter مع أكبر الآبار لاستخدامها. وأشار القيم في هذا البروتوكول قد تم اختيارها بعناية على أساس المقارنة الإحصائية لظروف مختلفة اختبارها. الميزة الأكثر إثارة للدهشة من هذا الاختبار هو على الأرجح أنه يمكن الاحتفاظ C.elegans في 96 لوحات جيدا أن ومختومة بالشمع. لم جنبا إلى جنب مقارنات لم تكشف عن أي اختلاف في عمر الحيوانات المستزرعة في لوحات تفض ، في لوحات مختومة مع السداده البلاستيك ، أو في لوحات مختومة مع السدادات التي تتيح تبادل الهواء. في جميع الحالات الثلاث ، وضعت الحيوانات متجانس جدا من L1 حامل للبالغين في غضون 65 ساعة وأظهرت أعمار مماثلة للغاية. وضعت الحيوانات نمت بالتوازي على NGM أسرع قليلا ، ولكن هذا الاختلاف كان مستقلا من غياب أو وجود السداده. ويستند البروتوكول عرضت على البكتيريا الحية ، ولكن يمكن تكييفها للبكتيريا ميتة. ومع ذلك ، في فحص السائل يمكن لبكتيريا واحدة على قيد الحياة تتكاثر بسرعة وإعادة ملء الثقافة. في أيدينا ، والطريقة الوحيدة التي يعول عليها لقتل البكتيريا إلى حد أنه يمكن استخدامها للثقافة السائل هو عن طريق العلاج لفترات طويلة من البكتيريا مع أشعة غاما. نقطة واحدة من المهم النظر في التخطيط لتجربة عمر هي قوة الكشف. اعتمادا على حجم التأثير ، لا بد من اختبار تأثير المخدرات من اهتمام في آبار متعددة. في مقايسة 4 نموذجية للأدوية المعالجة ومراقبة الآبار 4 ، الموافق 40-50 تقريبا كل الحيوانات ، يجب أن يكون كافيا للكشف عن زيادة بنسبة 30 ٪ في عمر في أكثر من 95 ٪ من التجارب. يتم الكشف عنها إلا زيادة قدرها 14 ٪ في 60 ٪ من الحالات ، وبالتالي تتطلب المزيد من الآبار تكرار. ويمكن استخدام ثروة من البيانات التي تم إنشاؤها بواسطة عمر هذا الاختبار لتطوير تجربة أو سلالة معينة من النماذج إن السفارة parametrical 1. هذه النماذج إن السفارة هي مفيدة لتحديد قوة الكشف وتقدير عدد من ايجابيات وسلبيات كاذبة على نطاق واسع الشاشات. علينا التحقق من التوقعات من هذه النماذج في التجارب الأعمى واستخدموه لتقدير أعداد من السلبيات كاذبة في شاشات كبيرة (نتائج غير منشورة). باختصار ، نحن نتوقع أن يقدم مقايسة سيكون ذات فائدة كبيرة لتحديد الجزيئات الصغيرة التي تمتد من عمر C.elegans ودراسة الآليات الكامنة.

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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measuring caenorhabditis elegans life span in 96 well microtiter plates

عمر هو عملية بيولوجية التي تنظمها عدة مسارات الوراثية. استراتيجية واحدة للتحقيق في بيولوجيا الشيخوخة في دراسة الحيوانات أن الطفرات في مكونات الميناء من هم في سن التنظيمية المسارات. إذا كانت هذه الطفرات التشويش وظيفة مسار العمر التنظيمية وبالتالي يغير عمر الكائن الحي بأكمله ، لأنها توفر رؤى الآلية الهامة 1-3. استراتيجية أخرى للتحقيق في تنظيم عمر هو استخدام جزيئات صغيرة لمسارات العمر التنظيمية التشويش. حتى الآن ، وتعرف على عدد من الجزيئات لتمديد عمر الكائنات الحية في نموذج مختلف ، وتستخدم كأدوات لدراسة بيولوجيا الشيخوخة 16/04. عدد من الجزيئات التي تم تحديدها حتى الآن هو صغير مقارنة مع &quot;مجموعة أدوات&quot; الجينية التي تتوفر لدراسة بيولوجيا الشيخوخة. ايليجانس انواع معينة هي واحدة من حيث المبدأ النماذج المستخدمة لدراسة الشيخوخة بسبب الوراثة الممتازة والعمر قصير من ثلاثة أسابيع. في الآونة الأخيرة ، برز بوصفه الكائن C.elegans نموذجا للشاشات النمط الظاهري يستند 5،7،16-20 المخدرات بسبب صغر حجمها وقدرتها على النمو في لوحات microtiter. نحن هنا تقديم مقايسة لقياس عمر C.elegans في 96 لوحات microtiter جيدا. كانت تجربة البلدان المتقدمة النمو واستخدمت بنجاح على شاشة كبيرة للمكتبات الجزيئات التي تمتد C.elegans عمر 7. وقد تم تقييم مدى موثوقية مقايسة في اختبارات متعددة : أولا ، عن طريق قياس عمر الحيوانات البرية نوع نمت في درجات حرارة مختلفة ، وثانيا ، من خلال قياس عمر المسوخ مع أعمار تغيير ، وثالثا ، من خلال قياس التغيرات في عمر ردا على تركيزات مختلفة من Mirtazepine المضادة للاكتئاب. Mirtazepine سبق ثبت لتمديد عمر في C.elegans 7. نتائج هذه الاختبارات تبين أن مقايسة غير قادرة على تكرار النتائج السابقة من فحوصات أخرى والكمي. تنسيق microtiter أيضا يجعل هذا الاختبار عمر الآلي متوافقة مع نظم مناولة السائل ، ويتيح الاندماج منصات الآلي.

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Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

في هذا البروتوكول نقدم طريقة لقياس<em&gt; انواع معينة ايليجانس</em&gt; عمر في 96 لوحات microtiter جيدا.

قياس انواع معينة ايليجانس الحياة سبان في 96 لوحات Microtiter حسنا

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. في هذا البروتوكول نقدم طريقة لقياس انواع معينة ايليجانس عمر في 96 لوحات microtiter جيدا.

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.

قياس انواع معينة ايليجانس سبان الحياة على وسائل الاعلام الصلبة

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.

قياس سبان الخميرة الحياة الزمني بواسطة خلايا ثمرة المسنين

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.

قياس تنسخي سبان الحياة في الخميرة الناشيء.

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.

الوقت الفاصل بين مرحلة التطور الجنيني المبكر من الميكروسكوب في انواع معينة ايليجانس

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.

الايس كاب : منهج في تزايد Arabidopsis الطماطم والنباتات في لوحات 96 - جيدا لالرفيع المناولة التنميط الجيني

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.

الأساسية<em&gt; Caenorhabditis ايليجانس</em&gt; طرق: التزامن والمراقبة

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.

قياس مقاومة الاكسدة من<em&gt; انواع معينة ايليجانس</em&gt; في 96-جيدا لوحات عيار مكروي

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.

قياس سمية الببتيد RAN في C. elegans

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.

استخدام Elegans Caenorhabditis إلى الشاشة لتفاعلات Chaperone الأنسجة محددة

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