这项工作是支持到A - LH从弥敦道冲击中心（P30 AG13283）和授予R01（R01 AG028516）从国家老化研究所（NIA）的试点奖

<html> 1。细菌浓度的估计建立校准曲线本节描述为任何给定的菌种（如OP50，HT115），将作为食物来源C.限制饮食实验使用细菌浓度的估算方法线虫 。每一株用于细菌，应建立单独的校准曲线。 <ol><li>接种单菌落的E.大肠杆菌 OP50细菌捡到3毫升的液体LB培养基中的细菌从一个新鲜的LB琼脂连胜。 </li><li>广场OP50文化在37℃摇床，并允许细菌的生长过夜。 </li><li>准备从过夜培养的菌悬液的序列的2倍稀释（2X，4X，8X，16X，32X）。 </li><li>一式三份分光光度计在600 nm测量吸光度（光密度），每个稀释度。 </li><li>进一步淡化每个菌悬液百万倍（两次稀释1000倍）。 </li><li>传播0.4毫升细菌悬浮液，每个稀释度100毫米的LB琼脂平板上均匀地。 </li><li> OP50在37℃培养箱中培养18-24小时发生的LB平板。 </li><li>计数每盘上生长的细菌菌落的数量。用于计算每个样本菌落计数每毫升CFU（菌落形成单位）。 </li><li>测OD 600值和OP50细菌浓度之间的关系可以建立绘制外径 600细菌浓度（CFU / ml为）的功能，并执行线性回归分析（图1）。 </li></ol>解决方案： <table border="1"><tbody><tr><td colspan="2">卢里亚肉汤（LB），1L： </td></tr><tr><td > 10克</td><td>细菌用的蛋白胨</td></tr><tr><td> 5克</td><td>酵母提取物</td></tr><tr><td> 10克</td><td>氯化钠</td></tr></tbody></table>加入去离子水至1L。 高压灭菌器，并在室温下储存。 2。固体板为主的饮食限制（SDR）板的制备本节介绍了编制固体板为主的饮食限制（SDR）的实验中使用的板材。通常情况下，我们认为细菌浓度为1 × 10 11 CFU / ml为自由采食的饲养条件和1 × 10 8 CFU / ml为最优的DR这种方法喂养的条件。然而，4-6浓度不同的细菌，可能需要建立一个完整的剂量依赖关系，SDR和它的响应。 <ol><li>接种单菌落的E. 3毫升液体LB培养基中的大肠杆菌OP50细菌。 </li><li>放置在37℃摇床OP50文化，使细菌的生长为6-8小时。 </li><li> OP50文化转移到一个新的烧瓶中含500 mL LB培养基中，放置在37℃摇床OP50文化过夜。 </li><li>测量外径600 OP50文化，以确定细菌浓度（使用第1部分中所产生的校准曲线的浓度计算） 。吸光度的准确测量，可能需要适当稀释。 </li><li>佩莱OP50细菌离心20分钟，在1500克。 </li><li>浓度1 × 10 12 CFU / ml为重悬细菌。 </li><li>准备在6个不同浓度10毫升细菌文化：1 × 10 12 10 11 10 10 10 9 10 8 10 7 CFU /毫升。 </li><li>加入0.2 mL的细菌培养成一个60毫米的中心凝固NGM的板块。避免接触枪头板的表面，表面上的刻痕允许进入琼脂平板蠕虫洞穴。 </li><li> 1小时37 °彗星孵化器板。 </li><li>将10μL的100mg/mL羧和10μL50mg/mL卡那霉素，每块板，以阻止细菌的生长。 </li><li>商店板与不同浓度抗生素杀害细菌在4 ° C以供将来使用（一个月）。重要的是整个计划实验的准备和储存足够板提前至少1-2天。使用同一批次的整个实验板是可取的。 </li><li>要验证的细菌的生存能力，刮去一些细菌的SDR板和传播与羧苄青霉素和卡那霉素的空NGM板。板放置在37 °彗星孵化器为6-8小时，然后检查细菌的生长（无细菌生长，应遵守）。 </li></ol>解决方案<table border="1"><tbody><tr><td colspan="2">线虫的生长介质（NGM），1L： </td></tr><tr><td > 3摹</td><td>氯化钠</td></tr><tr><td> 2.5摹</td><td>细菌用的蛋白胨</td></tr><tr><td> 20克</td><td>洋菜</td></tr></tbody></table>ove_content“&gt;高压灭菌40分钟，放凉至65 ° C，然后添加： <table border="1"><tbody><tr><td > 1毫升</td><td> 1个M硫酸镁4 </td></tr><tr><td> 1毫升</td><td> 1个M氯化钙2 </td></tr><tr><td> 1毫升</td><td> 5 mg / mL的胆固醇</td></tr><tr><td> 25毫升</td><td> 1M磷酸钾，pH值6.0 </td></tr></tbody></table>立即倾注到60毫米× 15毫米培养皿在10毫升分装液体NGM 3。设置SDR实验对于大多数的SDR相关的研究，如寿命分析和生育率分析，一个时代同步的蠕虫病毒的人口的准备，是至关重要的。对于其他研究中，简单地转移从一个普通的OP50板的蠕虫SDR板开始限制饮食。在本节中，设立SDR动物寿命分析的协议被描述为一个例子。 <ol><li>使用铂金蠕虫选择器转移OP50 20-30繁殖到几个经常NGM的板块活跃的成年人。根据实验所需的鸡蛋的数量，需要更多的成年人可能。 </li><li>离开板块在20 ° C为4个小时，让产蛋。 </li><li>从盘子中取出的成年人。板材应目视检查鸡蛋。 </li><li>离开板块在20℃，让后代发展成Late-L4/day 1成人阶段。通常，这需要3天时间为N2野生型蠕虫病毒在20 ° C。 </li><li> SDR板转移到适当数量的第1天成人开始限制饮食。 </li><li>寿命分析，建立了6-8板，每盘10-20蠕虫每个SDR条件。 </li><li>分数每个蠕虫的生存能力，每2天，直到所有的蠕虫已经死亡。至关重要的是，蠕虫病毒被转移到新鲜SDR板，每12-48小时，以避免食物完全剥夺。 </li></ol> 4。代表性的成果： 在图1所示是一个外径600 OP50在浓度估计为代表的校准曲线图。通过线性回归分析方程，可用于未来OP50细菌浓度计算。在图2所示是化验慢性SDR治疗效果上不同的蠕虫病毒株的平均寿命的一个例子。蓝线代表正常的野生型蠕虫特别提款权的剂量依赖性反应。红线表示，部分由DRR - 2 OE抑制SDR寿命的影响，而DAF - 16（mu86）并没有对SDR -诱导寿命延长的效果。执行C.寿命分析的详细方法线虫是在以往的文献1-3。 <img src="/files/ftp_upload/2701/2701fig1.jpg" alt="图1" /> 代表性的结果为E 的估计校准曲线图1。 大肠杆菌 OP50浓度。 OP50浓度绘制测OD 600值的函数。误差棒= 1.5X，2X，4X，8X，16X，32X的SD隔夜细菌培养前测吸光度稀释，CFU测量前进一步稀释1000倍的两倍。 <img src="/files/ftp_upload/2701/2701fig2.jpg" alt="图2" /> 图2 A C.代表性的结果。 线虫寿命的实验比较野生型N2动物（蓝色）特别提款权（青等。图4）与两个不同突变（绿色和红色）。误差线=扫描电镜

<ol>
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T., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+aging+and+age-related+disease+by+DAF-16+and+heat-shock+factor.">Regulation of aging and age-related disease by DAF-16 and heat-shock factor.</a> Science. 300, 1142-1145 (2003).</li><li>Kenyon, C., Chang, J., Gensch, E., Rudner, A., Tabtiang, R. <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+mutant+that+lives+twice+as+long+as+wild+type.">A C. elegans mutant that lives twice as long as wild type.</a> Nature. 366, 461-464 (1993).</li><li>Apfeld, J., Kenyon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lifespan+by+sensory+perception+in+Caenorhabditis+elegans.">Regulation of lifespan by sensory perception in Caenorhabditis elegans.</a> Nature. 402, 804-809 (1999).</li><li>Ching, T. 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饮食限制（DR）是已知的，以增加寿命和延迟在广泛的物种，包括哺乳动物5-7中各种与年龄有关的的疾病的发病。已用于环境和遗传操作模式在 C DR 线虫，包括在定义一个半液体介质中8,9，稀释10-12日在液体介质中的细菌的食物来源和使用行为的突变体，饲料以较慢的速率13培养蠕虫。然而，DR的遗传模拟可能产生的表型无关的DR，而在液体培养基中培?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>羧</td><td> Duchefa Biochemie </td><td> C0109.25 </td><td>溶于水。 </td></tr><tr><td>卡那霉素</td><td>罗氏公司</td><td> 10106801001 </td><td>溶于水。 </td></tr><tr><td>细菌用的蛋白胨</td><td>流式细胞，Dickinson公司</td><td> DF0118072 </td><td></td></tr><tr><td>细菌用的蛋白胨</td><td>流式细胞，Dickinson公司</td><td> DF0123075 </td><td></td></tr><tr><td>细菌用的酵母提取物</td><td>流式细胞，Dickinson公司</td><td> DF0127071 </td><td></td></tr><tr><td>洋菜</td><td>流式细胞，Dickinson公司</td><td> DF00145170 </td><td></td></tr><tr><td>无菌培养管</td><td>美国科学</td><td> 1485-0810 </td><td></td></tr><tr><td>分光光度计</td><td> Amersham公司生物科学</td><td>基因定量亲</td><td> Amersham公司是通用电气医疗集团的一部分。 </td></tr><tr><td>解剖显微镜</td><td>蔡司</td><td> STEMI 2000 </td><td></td></tr><tr><td>离心分离</td><td> Beckman Coulter公司</td><td> Accuspin FR </td><td></td></tr></tbody></table>

solid plate-based dietary restriction in caenorhabditis elegans

没有营养不良或饥饿，减少食物的摄入，增加寿命和延迟发病的各种与年龄有关的疾病，在广泛的物种，包括哺乳动物。这也导致在体重和生育能力下降，以及血浆葡萄糖，胰岛素和IGF - 1在这些动物的较低水平。这种治疗方法通常被称为饮食限制（DR）或热量限制（CR）。 线虫已经成为一个重要的模式生物，研究衰老的生物学。环境和遗传操作已被用于模型的DR，并已显示出在 C的寿命延长线虫 。但是，许多报道DR 在 C的研究线虫是通过在液体介质中传播的动物，而在老龄问题领域的遗传研究的最标准在固体琼脂培养皿板。在这里，我们目前使用标准的固体NGM杀害细菌的琼脂板DR的协议。

Watch this Scientific Journal Video about Solid Plate-based Dietary Restriction in Caenorhabditis elegans at JoVE.com

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

在这里，我们目前执行固体板为主的饮食限制在一个协议<em&gt; C。线虫</em&gt;与杀害细菌。

实心板为基础的饮食限制在线虫</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 ...

solid-plate-based-dietary-restriction-in-caenorhabditis-elegans

Research

JoVE Journal

Biology

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

16.5K 次观看.  University of Michigan. 在这里，我们目前执行固体板为主的饮食限制在一个协议 C。线虫与杀害细菌。

视频: Solid Plate-based Dietary Restriction in Caenorhabditis elegans

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.

测量线虫固体介质的寿命

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

科研

生物学

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

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

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.

时间推移显微镜早期胚胎发育线虫

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

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.

基本的<em&gt;线虫</em&gt;方法：同步和观察

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.

前列腺素的提取与分析<em&gt;线虫</em

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

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

多不饱和脂肪酸的膳食补充剂<em&gt;秀丽隐杆线虫</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

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.

线虫的耗氧率测定

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

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

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.

建模年龄相关的神经退行性疾病在 卡诺哈布迪蒂斯埃利根

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

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

自动化秀丽隐杆线虫的聚集定量

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...