我们感谢詹姆斯·C.克拉克为动物提供的数字，在不同的发育时间点。 的菌株在这项研究中获得的秀丽隐杆线虫的遗传学中心，这是由美国国立卫生研究院国家研究资源中心（NCRR）的支持。这项工作是从纽约市立大学支持的CIRG 1817 JL和惩教署和美国国立卫生研究院1R15GM073678-01和1R15GM097692-01，惩教署，我们感谢William水稻博士，博士娜塔莉亚·霍尔茨曼，梅丽莎Silvestrini的手稿上的评论。这项工作是进行在部分履行一个博士学位研究生中心纽约市立大学（S.雄）"的要求。

1。准备携带目的基因序列的RNA干扰进料板<ol><li>如果使用市售的RNAi库（ 例如，从源生物科学生命科学），继续执行步骤1.4。另外，克隆到载体L4440，一种常用的蠕虫RNAi质粒8的靶基因序列，通过标准的克隆协议。 </li><li>变换的菌株HT115（DE3）中，用25μg/ ml的羧苄青霉素的LB琼脂平板上培养和12.5微克/毫升四环素的重组质粒转化，并挑选单个克隆，以备将来使用。 </li><li>同时，转换到菌株HT115使用的实验作为对照的空载体L4440。 </li><li>文化重组和空L4440携带的细菌在1毫升LB肉汤与100μg/ ml氨苄青霉素过夜，在37°C。四环素还没有添加由于事实，即它减小RNAi效率。 </li><li>添加另一5毫升隔夜丘尔到100μg/ ml氨苄青霉素的LB肉汤重，另一个孵育4-6小时，在37°C。 </li><li>种子0.5毫升重组或空L4440携带细菌培养到的RNAi蠕虫板。标记所有板与克隆名称。 </li><li>马克板孵育过夜，在37℃至生长细菌草坪;这些的RNAi靶基因序列的带或不带板。至少有2块板应为每个条件制备。 </li></ol> 2。文化蠕虫的RNAi送料板<ol><li>火焰消毒，铂丝的尖端蠕虫挑。使用WORM挑来挑去6-10第四幼虫期（L4）雌雄同体每个RNA干扰板，重组或空L4440的动物将使用细菌作为食物来源。 </li><li>让雌雄同体的增长，在20°C（一个标准的文化条件线虫 ）过夜，他们将成为年轻的成年人的第二天。 </li><li> 6-10青壮年转移到一个新的相应标记，并准备RNA干扰板。 </li><li>让成人产卵4-6小时，然后取出所有的成年人板，这一步是同步的后代。一旦母亲被删除，开始计时。这是时间为零。 </li><li>将培养板在20°C，让动物长到特定的发展阶段，在该协议中，我们选择了年轻的成年人表型分析。击倒在一个正常的速度发展，培养72小时是足够的动物成为年轻的成年人。然而，不同的基因影响动物的不同发展。如果RNAi技术使动物生长速度不同，年轻人可以被认定为不超过24小时过去L4完成外阴的发展和2-6胚胎在子宫内（育）。要确定其他发展阶段，研究人员应使用性腺及外阴的发展为导向。 </li></ol> 3。分数车身尺寸表型的RNAi治疗的蠕虫<ol><li>将两层颜色标签磁带在载玻片上。使两个本身载玻片上。然后，将一个新的载玻片上，在两者之间的幻灯片用胶带。熔体在水中的2％琼脂糖;应用一个下拉到中心的新的载玻片上，然后按下顶端的第二玻璃上滑动，使一层薄薄的2％琼脂糖如前所述9。 </li><li>琼脂糖凝固后，取出顶部的载玻片上，，琼脂糖凝胶垫的幻灯片已经准备好加载蠕虫。标签幻灯片与克隆名字。 </li><li>加入10微升25毫米NaN的3琼脂糖垫;选择30-40动物的NaN 3解决方案，以固定，然后把盖玻片的顶部，重复重组和空L4440携带的RNA干扰治疗的动物。 </li><li>在解剖显微镜下的图像的蠕虫使用2.5倍的物镜，这里我们使用徕卡数码相机与支持软件Qcapture的，。 </li><li>校准，第一次拍摄图像的标准千分尺尺。然后打开图像的Image-Pro软件中，点击"测量"，然后选择"校准"，然后选择"空间校准向导"。随着"校正活动图像"选中，单击"下一步"。输入的名称的校准和确保的空间参考单元被设置到微米。然后，检查"创建参考校准"按钮，然后单击"下一步"。点击"参考线"。会出现一个参考比例尺。重新放置比例尺的千分尺的端部相匹配，则表明参考表示1,000个单位。点击"确定"，"下一步"，"完成"。 </li><li>一旦软件校准，打开一个蠕虫图像。然后，选择"测量"菜单下，选择"校准"，然后点击"选择空间"。在下拉菜单中，选择创建的文件名千分尺校准。然后，在"测量"菜单中，选择"测量"。在打开的窗口中，选择"自由绘制工具，并通过动物的身体，从头部到尾部与电脑鼠标（ 图1）的中心跟踪线。将在窗口的长度。现在你有两组数据:体长，治疗靶基因的RNAi和处理空L4440矢量的控制动物的动物。 </li><li>导出到Microsoft Excel文件，或其他合适的统计软件的长度测量。的数据进行分析，通过计算每个样本的平均值和标准偏差。然后比较学生的t检验的样品。 </li></ol>

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没有利益冲突的声明。

在这个协议中，我们描述的车身尺寸有缺陷的突变体C.识别方法通过RNA干扰喂养的线虫 。此方法是适用于TGF-β信号分量的识别。由于这样的组件通过进化15是高度保守的，这样的屏幕是在所有后生动物阐明的分子机制的TGF-β信号有关。在这样的屏幕设计是一个重要的考虑出发菌株。我们已经证明，无论ERI-1，LIN-15b和RRF-3更敏感的RNAi喂养比对照菌株，这与以往的研究12,13是一致的。然而，即使蓖麻-升;的lin-15b的表现出最强的表型，动物没有生长良好，并生产了少量的后代。因此，在一个大型的屏幕，我们不会有足够多的动物，得分从该菌株的表型。作为结果，我们主张RRF-3株申请RNA干扰喂养技术车身尺寸调节。考虑出发菌株的第二个选择是是否启动屏幕DBL-1通路完整或致敏株，其中DBL-1途径是妥协，或过度活跃的开始。这些可供选择的出发点都是可能导致的重叠，但不同的信号元件的识别。我们的协议也可以被修改，以研究其他胚后的表型。在这种情况下，感兴趣的表型的可得的动物的阶段，可能需要进行修改，通过改变在步骤2.5的增长的持续时间。启动其他表型的大型屏幕前，我们会建议试点屏幕产生兴趣的表型的基因，如我们在这里描述DBL-1，SMA-2，SMA-SMA-4和SMA- 6。 通过使用此协议，撞倒DBL-1通路元件表现出预期的体长型。 Ţ软管RNA干扰动物与对照组动物相比，长度约68-95％。在以前的研究中，DBL-1通路失去功能的基因突变在成年缩短约50％，比野生型动物7,10,11。因此，这里介绍的供给的RNAi技术并没有完全消除基因的活性。因此，该方法可以被限制在其能力，以确定有一个弱的表型的通路的组件。同时，由于有许多基因，调节人体的长度，从屏幕上的基因也可能不一定下降DBL-1途径。进一步的实验应进行候选人的途径，是真正的任何其他屏幕的方法。 总之，RNAi技术的喂养屏幕C.线虫已被证明可用于确定在感兴趣的过程中所涉及的基因。在这个协议中，我们优化了现有协议的机身尺寸为缺陷的识别是非常有效的。可以进一步优化的协议修改为其他胚后的表型的研究。

<table border="1"><tbody><tr><td></td><td></td><td></td><td> - RNA干扰蠕虫板 17.7克WORM介质混合 60克琼脂 H 2 O 3升。高压灭菌。 加入3毫升1M IPTG（无菌）和3毫升25毫克/毫升羧苄青霉素（无菌）;然后倒入培养皿60毫米。 蠕虫板 17.7克WORM介质混合 0.6克硫酸链霉素 60克琼脂 H 2 O 3升。高压灭菌。倒成60毫米的培养皿中。 WORM介质混合 的55克Tris-CL 的24克Tris-OH 310克细菌蛋白胨 800毫克胆固醇 200克氯化钠 调匀，一定要避免大块，可以存放许多个月前使用这种粉状混合物。 2％琼脂糖 0.5 G琼脂糖 广告D 25毫升蒸馏水2 O。在微波加热，直至溶解。 1M异丙基β-D-1-硫代吡喃半乳糖苷（IPTG）诱导 2克IPTG 毫升蒸馏水溶解IN10 2 O 1M NaN的3 6.5克楠3 添加蒸馏水至100毫升2 O 25毫米楠3 2.5毫升1M南3 添加蒸馏水至100毫升2 O 秀丽隐杆线虫线虫遗传学中心 L4440质粒（携带氨苄青霉素抗性基因） 细菌菌株HT115（DE3）中（包含经IPTG诱导T7聚合酶; RNA酶III基因（dsRNAse）也携带四环素抗性基因的缺陷）16 60毫米陪替氏培养皿 100毫米培养皿 14毫升的园底猎鹰文化管 玻璃幻灯片 盖玻片 1-20微升移液器 20-200微升移液器 200-1000微升移液器 1-200μL枪头 200-1000微升枪头 1.5毫升的Eppendorf管 铂丝蠕虫挑</td></tr></tbody></table>

using rna-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode c. elegans

双链RNA介导的干扰（RNAi）是一种有效的策略，以击倒靶基因的表达1-3。它已被应用到许多模型系统，包括植物，无脊椎动物和脊椎动物。有各种各样的方法来实现RNAi 在体内 4,5。例如，可以与靶基因的RNAi载体转化，然后永久或瞬时转化的细胞系或初级细胞实现的基因敲除效果，或者从特定的靶基因（RNAi的寡核苷酸）的合成的双链寡核苷酸，可以是瞬时转化靶基因沉默的细胞系或原代细胞或合成的双链RNA分子可显微注射到生物体。由于线虫C.的利用细菌作为食物来源，饲养的动物与对靶基因的表达双链RNA的细菌提供了一个可行的策略。在这里，我们提出的RNAi输送机身尺寸表型的方法来得分。车身尺寸C.线虫主要受型TGF-β -等配位体DBL-1，所以该法是适合识别的TGF-β信号元件7。我们使用不同的菌株，其中包括两个RNA干扰过敏株重复供给的RNAi实验。我们的研究结果表明，RRF-3株给了我们最好的预期的RNAi表型。该方法易于操作，重现性好，易于量化。此外，我们的协议，最大限度地减少了使用专门的设备，所以它是适合于规模较小的实验室或主要是本科院校。

在我们的研究中，我们专注上机身尺寸调节的DBL-1/TGF-β途径。 DBL-1途径体型小，更短的车身长度相比，与野生型动物7,10,11功能的损失。因此，屏幕为C.线虫的机身尺寸突变体能够确定TGF-β信号组件和改性剂。 DBL-1信号是介导的一个保守的TGF-β的信号转导途径，其中包括细胞表面受体与细胞内的Smad信号传感器12。要测试的有效性的RNAi通过喂养的车身尺寸突变体的识别，我们使用了这种技术击倒DBL-1通路组件:dbl-1/ligand; SMA-2，SMA-3，sma-4/Smads，;的sma-6/receptor。我们的研究中，我们使用了两种不同的RNA干扰过敏C.的菌株进行实验:ERI-1 LIN-15B RRF-3和13,14。同时，我们也用N2（STAndard野生型）菌株和lin-36，LIN-15途径的RNA干扰的敏感性，但类似N2 13的一个组成部分。我们的研究结果显示（ 图2），所有这些菌株如预期，显示短机身尺寸型（P &lt;0.001），SMA-6 RNAi在LIN-36的背景。 RRF-3的背景，年轻的成年人RNAi治疗后体长分别为84％〜95％单独的载体。在ERI-1的LIN-15B背景的年轻人，体长RNAi后的68〜86％，对照组动物。相比的lin-36和N2菌株，两者的RNAi过敏性菌株RRF-3和蓖麻-1;的lin-15b的更敏感。 <img alt="图1" src="/files/ftp_upload/4373/4373fig1.jpg" /> 图1。代表图像的动物身体长度的测量。A.测量前动物形象; <stroNG&gt; B.动物体的中心在测量过程中跟踪，跟踪线沿线动物的身体从头部到尾部，是衡量软件。 <img alt="图2" src="/files/ftp_upload/4373/4373fig2.jpg" /> 图2。 DBL-1通路组件已通过RNAi喂养方法灭活动物的体长。RNA干扰动物，所有这些背景株中显示短的车身长度预期的表型（P &lt;0.001），除SMA-6 RNAi在LIN-36背景。 RNAi的RRF-3和ERI-1-15B林株表现出了更强的表型的lin-36和N2背景。 <img alt="图3" fo:content-width="4in" fo:src="/files/ftp_upload/4373/4373fig3highres.jpg" src="/files/ftp_upload/4373/4373fig3.jpg" /> 图3。原理图ðescription的，利用RNAi喂养策略，以筛选候选基因。

Watch this Scientific Journal Video about Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans at JoVE.com

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

我们演示了如何使用供给的RNAi技术敲除目标基因和分体尺寸型<em&gt; C.线虫</em&gt;。这种方法可用于大规模的画面，以找出潜在的基因成分，如在车身尺寸上的DBL-1/TGF-β信号调节。

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

using-rna-mediated-interference-feeding-strategy-to-screen-for-genes

Nanyang Technological University

Research

JoVE Journal

Biology

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

12.3K 次观看.  Borough of Manhattan Community College, City Universtiy of New York (CUNY). 我们演示了如何使用供给的RNAi技术敲除目标基因和分体尺寸型 C.线虫。这种方法可用于大规模的画面，以找出潜在的基因成分，如在车身尺寸上的DBL-1/TGF-β信号调节。

视频: Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

科研

生物学

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用"冻结开裂"

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

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

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

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

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

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 ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

测量水润谷胱甘肽诱导饲养响应

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here protocols are presented for preparing embryos and adult C. elegans animals for short- and long-term time-lapse microscopy and methods for tracking and quantification of developmental processes. The methods presented are all based on C. elegans strains available from the Caenorhabditis Genetics Center and on open-source software that can be easily implemented in any laboratory independently of the microscopy system used. A reconstruction of a 3D cell-shape model using the modelling software IMOD, manual tracking of fluorescently-labeled subcellular structures using the multi-purpose image analysis program Endrov, and an analysis of cortical contractile flow using PIVlab (Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB) are shown. It is discussed how these methods can also be deployed to quantitatively capture other developmental processes in different models, e.g., cell tracking and lineage tracing, tracking of vesicle flow.

Tracking and Quantifying Developmental Processes in C. elegans Using Open-source Tools

Here it is shown how to track and quantify developmental processes in C. elegans. The methods presented are based on open-source tools that can be easily implemented. It is demonstrated how to reconstruct 3D cell-shape models, how to manually track subcellular structures, and how to analyze cortical contractile flow.

跟踪和量化的发育过程<em&gt;℃。线虫</em&gt;使用开源工具

Quantitatively capturing developmental processes is crucial to derive mechanistic models and key to identify and describe mutant phenotypes. Here ...

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.

测量在C.elegan的RAN肽毒性

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

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略

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此视频中的章节

一个线虫多巴胺神经元退化含量的应用验证潜在的帕金森氏症基因

核糖体束缚新生多肽的分离

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

抗体在染色

在大规模的基因敲除

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

方法来评估肌肉的亚细胞中

测量水润谷胱甘肽诱导饲养响应

跟踪和量化的发育过程

测量在C.elegan的RAN肽毒性