Name: measuring ran peptide toxicity in c. elegans
Uploaded: 2020-04-30T15:00:00
Description: Watch this Scientific Journal Video about Measuring RAN Peptide Toxicity in C. elegans at JoVE.com

NIH R21NS107797

1. 生成共性多变的 RAN 肽表达构造
<ol><li>利用同义词子共体设计单个 RAN 肽编码序列，以消除基本的重复 DNA/RNA 结构，但保留覆盖氨基酸序列。</li>
	<li>按研究所需的重复长度（通常为 5-100 个重复）以商业方式订购自定义协当序列。在 5' 末端包括 HindIII 限制站点和 3' 末端的 BamHI 限制站点，以方便克隆到C. elegans表达式载体，如 pPD95.79。 注：如果大型构造的合成证明困难，则可以合成较?...

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在这里，我们报告的方法，可用于测定RAN肽毒性建模在肌肉或神经元的C.elegans。虽然神经退行性蛋白质在人类患者中具有年龄发病表型，但在模型系统中过度表达时，它们也会表现出发育毒性。过度表达有显著的解释性限制，但它也为基因或药理学屏幕提供了一个强有力的起点，旨在识别能够逆转有毒表型的基因或药物。这一点尤其重要，因为大多数精确的动物疾病模型要么没有表型，要?...

DNA重复序列的不当扩展是几种神经退行性疾病的遗传基础，如肌萎缩性侧索硬化症（ALS）、前额痴呆症（FTD）和亨廷顿舞蹈症（HD）1。虽然这些疾病已建立细胞和动物模型，但这些条件背后的机制没有明确界定。例如，HD 是由亨廷顿蛋白 Htt2的编码序列中 CAG 重复序列的扩展引起的。由于CAG编码氨基酸谷氨酰胺，CAG重复膨胀导致在Htt内插入多谷氨酰胺或聚Q序列。 膨胀的聚Q蛋白形成长度和年龄相关蛋白聚合体，与毒性33，44相关。令人惊讶的是，最近的两项研究表明，聚Q序列的长度不是导致HD疾病发病的主要原因，这表明与多Q无关的因素也可能导致这种疾病55，6。6
一种可能的聚Q无关机制涉及一种新发现的蛋白质翻译类型，称为Repeat A分离N对AUG依赖（RAN）翻译7。顾名思义，RAN 翻译仅在存在扩展重复序列且不需要规范启动子序时发生。因此，RAN转换发生在重复的所有三个读取帧中，以产生三个不同的多肽。此外，由于许多基因还产生一个反感觉转录，其中包含扩展重复序列的反向补充，RAN翻译也发生在反意义笔录的所有三个阅读帧中。RAN 翻译共同将从扩大的含重复性 DNA 序列中产生的蛋白质数量从一个肽扩展到六种肽。迄今为止，RAN翻译已经观察到至少8个不同的重复膨胀障碍8。RAN肽在验尸病人样本中观察到，并且仅在患者进行扩大的重复99，1010的情况下。虽然这些肽在患者细胞中明显存在，但它们对疾病病理生理学的贡献尚不清楚。
为了更好地确定与RAN肽相关的潜在毒性，几个小组在各种模型系统中表示每种肽，如酵母、苍蝇、小鼠和组织培养细胞11、12、13、14、15、16。11,12,13,14,15,16这些模型没有利用重复序列进行表达，而是采用一种共变法，即消除重复序列，但保留氨基酸序列。翻译启动通过规范的ATG发生，肽通常融合到N-或C-terminus的荧光蛋白，这两种蛋白质似乎都不会干扰RAN肽的毒性。因此，每个构造都过度表示单个 RAN 肽。通过简单的测定对多细胞生物体中不同的RAN产品进行建模，以测量RAN肽毒性，对于了解不同RAN产品从每个致病的重复扩张如何导致细胞功能障碍和神经退化至关重要。
与其他模型系统一样，C. elegans提供了一个灵活而高效的实验平台，能够研究新的疾病机制，如 RAN 肽毒性。蠕虫提供了几种独特的实验属性，目前在其他RAN肽毒性模型中不可用。首先，从出生到死亡，C.elegans在光学上是透明的。这允许对RAN肽表达和定位进行简单的可视化，以及活体动物神经退化的体内分析。其次，生成RAN肽表达模型的转基因方法价格低廉，速度快。鉴于C.elegans的短三天生命周期，在一周内可以产生稳定转基因线，以细胞型特定的方式表达任何给定的RAN肽。第三，简单的表皮输出可以与基因筛选方法相结合，如化学诱变或RNAi筛选，以快速识别RAN肽毒性所必需的基因。最后，C. elegans的短寿命（+20天）使研究者能够确定衰老，这是大多数重复扩张性疾病的最大危险因素，如何影响RAN肽毒性。这种实验属性的组合在任何其他模型系统中都是无可比拟的，为RAN肽毒性的研究提供了一个强大的平台。
在这里，我们描述了几个测定，利用C.elegans的实验优势来测量RAN肽的毒性，并确定这种毒性的基因修饰剂。Codon-uns-ATG启动的RAN肽被标记与GFP，并在肌-3启动器下的肌肉细胞或非c-47启动子作用器下的GABAergic运动神经元中单独表达。对于肌肉细胞的表达，重要的是有毒的RAN肽被标记绿色荧光蛋白（GFP），或其他荧光蛋白（FP）标签，可以针对RNAi喂养载体。这是因为有毒的RAN肽表达通常阻止生长，使这种菌株不可行。使用gfp（RNAi）有条件地不激活RAN肽表达，并允许菌株维持，遗传交叉等。对于检测，这些动物从gfp（RNAi）中去除，这允许表达RAN肽和由此产生的表型。除了设计Codon-变型RAN肽表达构造的分子策略外，我们还描述了用于测量发育毒性（幼虫运动和生长测定）、发育后年龄相关毒性（麻痹测定）和神经元形态缺陷（共性测定）的测定。

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			<td>Microinjection to generate RAN peptide transgenic strains</td>
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			PETRI DISH, 60X15MM,500/CS</td>
			<td>CORNING LIFE SCIENCES PLASTIC</td>
			<td>FB0875713A</td>
			<td>Nematode growth plates and RNAi</td>
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			<td>TISSUE CULT PLATE 24WEL 50/CS</td>
			<td>CORNING LIFE SCIENCES DL</td>
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measuring ran peptide toxicity in c. elegans

C. elegans通常用于模拟由重复扩张突变引起的与年龄相关的神经退行性疾病，如肌萎缩性侧索硬化症 （ALS） 和亨廷顿舞蹈症。最近，含有重复膨胀的RNA被证明是一种新型蛋白质翻译的基质，称为重复相关非AUG依赖性（RAN）翻译。与规范翻译不同，RAN 翻译不需要启动协和，并且仅在重复超过阈值长度时发生。由于没有开始协理来确定读取帧，RAN 翻译出现在包含重复扩展序列的感知和反感觉 RNA 模板的所有读取帧中。因此，RAN 翻译将可能的疾病相关有毒肽的数量从 1 扩展到 6。到目前为止，RAN翻译已经记录在八种不同的重复扩张为基础的神经退行性疾病和神经肌肉疾病。在每种情况下，破译哪些RAN产品是有毒的，以及它们的毒性机制，是了解这些肽如何促进疾病病理生理学的关键步骤。本文提出了在模型系统C.elegans中测量RAN肽毒性的策略。首先，我们描述了测量RAN肽毒性的程序，对发育的C.elegans的生长和活力。其次，我们详细介绍了一种用于测量RAN肽在运动性上发育后、年龄依赖作用的测定方法。最后，我们描述了一种神经毒性测定，用于评估RAN肽对神经元形态的影响。这些测定提供了RAN肽毒性的广泛评估，可用于执行大规模遗传或小分子屏幕，以确定疾病机制或疗法。

我们使用此处描述的测定来评估不同基因抑制对在具有 G4C2重复扩张的 ALS 患者中发现的 RAN 二肽毒性的影响。利用生长测定测量发育毒性，我们分析了在全基因组RNAi屏幕抑制器中识别的几种基因敲除突变体对肌肉表达PR50-GFP毒性的影响。虽然仅表达PR50-GFP就导致完全渗透性生长抑制，但几个基因的功能突变的丧失抑制了PR发育毒性的12-94%（图1A）。
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Measuring RAN Peptide Toxicity in C. elegans

重复相关非ATG相关转化产品是几种重复扩张性疾病的病原特征。所述协议的目的是使用模型系统C.elegans的行为和细胞测定来评估这些肽引起的毒性。

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 ...

RAN肽是重复扩张性神经退行性疾病的一个特征，如亨廷顿舞蹈症、肌萎缩性侧索硬化症和前脑膜痴呆症。这些协议为C.elegans模型系统中的 RAN 肽毒性定量提供了一种重要的标准化方法。所述技术简单易学，价格低廉，可以快速执行，并利用C.elegans系统的可重复特性，如移动和复制。在尝试此协议时，主要的挑战是获得一致的 RAN 肽毒性和表达。控制的主要因素包括药物温度、温度变化的时间和检测板的准备。实验者必须在这个与年龄相关的过程检测中对动物的表型进行一致分类。该技术的可视化演示显示了它们的分类标准。首先，用一毫摩尔IPTG和每毫升25微克的碳素将标准线虫生长介质倒入24孔板中。将 HT115 细菌中的 RNAi 喂养到 LB 卡贝尼西林加糖上，并在 37 摄氏度下生长 24 小时。第二天，在250转/小时摇晃时，将单个菌落放入一毫升LB卡贝尼西林液体介质中，在37摄氏度下生长细菌18小时。如文本手稿所述，发现 NGM RNAi 24 孔板的四个单独孔，含有 20 微升的隔夜细菌培养物，并允许 RNAi 细菌在一夜之间诱导双链RNA生产。使用标准的次氯酸盐方法从表达综合 RAN 肽转基因的第一天成年 C.elegans 分离卵子。然后将大约30个卵子播种到24井板的每个井中，并在20摄氏度下孵育该板7天。要执行视频速度分析，使用装有单色摄像机的立体解剖显微镜，从两个单独的孔中获取两个视频，用于处理每个 RNAi 条件。确保在井之间使用一致的采集设置。对于每个视频，将持续时间设置为 10 秒，将时间间隔设置为 76 毫秒。将图像曝光时间设置为四毫秒，并使用 1.49 缩放设置。然后选择两个按两个装箱。验证分辨率为 800 x 600 像素并开始录制。采集后，立即用应变、RNAi 状况和井号标记每个视频。然后测量每个视频中 20 种不同动物的行驶距离和长度，检查每个 RNAi 条件的总共约 40 个蠕虫。在软件中，选择注释工具按钮。然后绘制文本工具。单击在视频的第一帧中测量的蠕虫中心一个点，然后用数字标记它。在直观地跟踪蠕虫的同时，将视频推进到末尾。然后单击它，然后用下一个相应的数字标记它。使用绘制比例条工具绘制一条从第一个数字到第二个数字的线，并在电子表格中记录行驶距离。对视频中的其他 19 个蠕虫重复此测量，同时在分析窗口中保留每个单独的测量值。测量完成后，拍摄最终视频帧的快照，说明所有测量线，以便数据可追溯到其来源的动物。接下来，使用四列电子表格分析数据，其中第 1 列是蠕虫标识符，第 2 列是速度。通过右键单击实验下的视频并访问属性，可以找到每个视频的时间。要使速度测量正常化，请找到每个动物的长度。使用绘制折线工具从头尖到尾部尖端自由跟踪 C.elegans。然后拍摄最终视频帧的快照，说明所有折线。行的长度将在统计下记录。在电子表格中添加两列，一列用于动物长度，另一列用于规范化速度。最后，使用单向 ANOVA 与空向量 RNAi 进行非正常测试分析规范化速度数据。将四到六个转基因L4 C.elegans在6厘米的GP RNAi板上放置表达 RAN肽GP，并在20摄氏度下生长。如果实验是利用RNAi来测试对RN肽毒性的遗传影响，则将10名转基因重力成人移动到两个空载体RNAi GFP RNAi或基因特异性RNAi板中。如果突变体被用来分析对 RAN 肽毒性的遗传影响，则将 10 种重力野生类型或突变动物在两个空载体 RNAi 或 GFP RNAi 板块上放置 10 种表达相同 RAN 转基因的突变动物。在20摄氏度下生长48小时。从 RNAi 板中挑选 10 组 10 个转基因 L4，将每组 L4 放在三厘米 RNAi 板上。确保为测定选择的蠕虫都有表面正常的活动性。将盘子放在塑料袋中，以保留水分，并在 25 摄氏度下孵育。24小时后，通过敲击解剖显微镜上的板检查运动，分析动物的机动性。如果蠕虫移动超过身体长度，将其计为移动，并转移到新的三厘米 RNAi 板。使用铂金拾取来敲击头部和尾部的剩余蠕虫。如果动物移动超过身体长度，将动物计数为移动，并转移到一个新的三厘米RNAi板。如果第二天空病媒控制中未观察到严重瘫痪，则终止测定并重新开始。将所有非移动蠕虫分组在一起，以便轻松检测超过体长的运动。数一数所有瘫痪、袋装或死蠕虫，然后丢弃盘子。继续每天给动物打分，持续五到七天。生长测定用于测量基因抑制对在具有G4C2重复扩张的ALS患者中发现的 RAN二肽毒性的影响。仅PR50 GFP的表达就导致完全生长停止，但几个基因的敲击抑制了发育毒性。利用视频速度分析法测量了特定基因敲击对PR50表达动物发育运动的影响。正如预期的那样，与空向量RNAi相比，PR50 GFP表达的抑制导致运动性显著增加。此外，RNAi对蛋白酶体亚单位RPN7导致PR50运动性显著增加。当用年龄依赖性麻痹测定分析成人表型时，PR50 GFP在五天前表现出高达80%的瘫痪。然而，RNAi针对基因cul-6显著延迟瘫痪，表明PR50 GFP毒性需要cul-6。为了确定 RAN 蛋白在 C.elegans 神经元中表达时是否引起神经病理学，使用小卖部测定法检查了神经元特异性毒性。在第二天成人，PR50 GFP表达在运动神经元导致显著增加的运动神经元出血。胰岛素IGF受体基因同源DAF-2的突变显著抑制了这种神经病理学，该突变延缓了几种神经退行性蛋白质的毒性。对于行为分析，RAN肽必须产生高度的佩内性表型。这种基因或药物可以为 RAN 肽疾病提供新的生物标志物或治疗选择。使用这些测定方法，我们为C9ORF72相关冉多肽精氨酸的抑制者进行了全基因组RNAi筛查。在这个屏幕中识别的基因包括许多新的和保存的基因，这些基因将成为未来机械研究的主题。

Research

JoVE Journal

Biology

Measuring RAN Peptide Toxicity in C. elegans

Generation of Stable Transgenic C. elegans Using Microinjection

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

测量线虫寿命在96孔板

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

C. elegans Chemotaxis Assay

C. elegans Chemotaxis Assay

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

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

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

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 ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...