Name: the replica set method: a high-throughput approach to quantitatively measure caenorhabditis elegans lifespan
Uploaded: 2018-06-29T14:00:00
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本手稿所描述的这项工作的经费由下列机构提供: 教务长大学院长办公室和医学院和牙科院长办公室, 通过卫生科学中心进行计算创新 (HSCCI);埃里森医学基金会新学者老龄化研究 (AG-NS-0681-10) 资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。

1. 传统的线虫寿命评分方法
<ol><li>试剂的制备
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<li>通过基于喂养的 rna 识别基因进行灭活。购买已转换的 HT115大肠杆菌2的股票, 其中含有对利益的 rna 克隆。另外, 亚克隆的基因 cDNA 的 multicloning 的 L4440 质粒的网站。 注: HT115 是一种 RNase III. 型大肠杆菌菌株, IPTG 诱导 T7 聚合酶活性, 用于防止细菌中 dsRNA 的降解。对于不使用基于喂养的 rna 干...

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传统和副本集方法都需要对年代衰老动物进行同步处理。我们包括一种方法, 使动物使用次氯酸钠治疗妊娠成人, 只有受精卵与妊娠成人生存治疗。这些胚胎孵化在液体悬浮和发育停止在第一幼虫阶段 (L1)。在将 L1 动物播种到食物上后 (如大肠杆菌表达 dsRNA 的基因), 动物恢复发育。用次氯酸盐治疗妊娠 L1 动物, 可以使残留的非种子选手 L1 动物在液氮或-80 摄氏度冷藏库中无限期地冷冻?...

在了解衰老的遗传基础方面, 最具变革性的技术进步之一是在1线虫中发展基于喂养的 rna 干扰;在实验性地使用 rna 干扰之前, 许多衰老的表型不是遗传的。以喂养为基础的 rna 干扰是通过生产大肠杆菌中的 dsRNA 与内源c. 线虫mRNA: IPTG 诱导双向转录跨插入的任何c. 线虫cDNA 或一部分的在质粒2中打开阅读框架。当C. 线虫饲料在完整的大肠杆菌,由细菌产生的 dsRNA 通过 SID-2 跨膜蛋白3从流明传输到肠道细胞, 然后通过 SID-14分布到其余的动物。在每个细胞内, 外源 dsRNA 由 Dicer 复合体处理成 siRNA, 通过互补基配对与成熟的 mRNA 相互作用, 创建一个新的 siRNA-mRNA 双工。这种双工是由 RISC 复合体和劈裂的, 从而降解内源 mRNA5。因此, 仅仅改变质粒的插入, 你就可以使几乎任何基因在线虫基因组中的功能失效。这一发现导致了几个大的基于喂食的 rna 干扰库的创建--转化的大肠杆菌种群的集合, 可以结合起来以达到约86% 已知的C. 线虫基因的覆盖率6,7。
自从饲养基 rna 干扰的推进, 在C. 线虫的全面屏幕导致发现了900多个基因, 改变生命的灭活时 (由 WormBase 的 rna 干扰-表型协会证明), 我们提到为 gerogenes。大多数 gerogenes 在长寿控制中的作用是在一些精算报告中发现的, 通过喂食为基础的 rna 干扰 (见图 1A和补充文件 1的细节)。在某些情况下, 这些 gerogenes 是根据在单个或几个时间点测量生存能力而确定的, 这不能为使用 rna 干扰治疗的寿命变化提供可量化的衡量标准。在其他情况下, 这些基因已经定量地评估了寿命的变化, 以及其他年龄相关的表型。例如, 我们以前确定了159种基因, 对正常和增加的动物寿命都是必要的, insulin/IGF-1 信号减少, healthspan 的量化变化。其中, 103 基因 inactivations 导致 progeric 表型, 因为损失导致一个或多个过早衰老的迹象8。
虽然有些 gerogenes 与100项或更多的研究相关 (例如 daf-16、daf-2、sir-2.1), 但超过 400 gerogenes 有10或更少的引文 (图 1B和补充文件 2)。因此, 虽然综合饲料基 rna 干扰屏幕发现和马虎的特点数以百计的假定 gerogenes, 如何这些基因在长寿控制中的作用, 这些基因产品之间的遗传关系仍然很差研究。对年龄相关表型的全纵向分析是确定 gerogenes 之间的基因相互作用 (如上位相互作用、asynthetic 相互作用等) 的先决条件。深入了解 gerogenes 之间的遗传相互关系需要一种高通量定量方法, 这也利用了基于喂养的 rna 干扰的优势。
衰老最常见的替代措施是寿命。传统的测量C. 线虫死亡率的方法是在一小部分人口样本中追踪个别动物的死亡情况。随着时间的推移, 动物数量相对较少, 并且周期性地用铂丝或睫毛轻轻地戳, 以运动作为生存能力的指示器 (图 2A)。这种方法得到了广泛的应用, 因为它提供了直接的平均值和最大寿命的测量。然而, 这种传统的方法是耗时和相对较低的吞吐量, 这限制了动物的数量和条件, 可以同时测量的控制方式。最近的一项模拟研究发现, 许多C. 线虫寿命研究并没有对足够数量的动物进行检测, 从而能够可靠地检测出条件9之间的细微变化。此外, 这种传统的方法包括反复处理同一队列动物随着时间的推移, 这反过来可以引入污染, 并可能损害或杀死越来越脆弱, 衰老的动物。
我们开发了一种替代的 "复制集" 方法来测量C. 线虫的寿命。为此, 大量人口的年龄同步, 等基因系动物被划分成少数人口 (或复制品)。生成的副本样本足以覆盖计划实验中的每个时间点。在每个观察时间点, 其中一个副本是为生存, 死亡和被截断的动物的数量得分, 然后动物在复制被丢弃。因此, 在整个人口的预期寿命内, 一系列独立的亚群定期取样 (图 2B)。在使用副本集, 没有重复的刺激动物, 并没有重复暴露在潜在的环境污染。在同一时间点观察到的生存能力完全独立于其他所有观察, 这将最大限度地减少处理和提高吞吐量。这使得我们能够定量8,10的上百个 rna 克隆的寿命变化。
在这里, 我们提出了通过复制集和传统的方法来进行c. 线虫寿命的详细协议, 以评分c. 线虫长寿。我们证明了相似的结果在方法之间得到。我们开发了软件, 以协助对通过任一方法生成的寿命数据进行图形分析, 我们可以根据 GPL V3 许可证免费提供这些信息 (见材料表)。"WormLife" 是在 R11中编写的, 包括图形用户界面 (GUI), 用于绘制数据, 该接口已在 Mac OS 和 Linux 中进行了测试。最后, 我们比较和对比了每种方法的局限性, 并强调在选择方法来测量线虫寿命的定量变化的其他因素。

<table border="0" cellpadding="0" cellspacing="0" width="1120">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">IPTG (isopropyl beta-D-1-thigalactopyranoside)</td>
			<td style="width:108px;">Gold Bio</td>
			<td style="width:119px;">12481C100</td>
			<td style="width:527px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">FuDR (5-Fluoro-2&#39;-deoxyuridine)</td>
			<td style="width:108px;">Alfa Aesar</td>
			<td>L16497</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:367px;">24 Well Culture Plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#662102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Retangular non-treated single-well plate, 128x86mm</td>
			<td style="width:108px;">Thermo-Fisher</td>
			<td style="width:119px;">242811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">600 &#181;L 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#786261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">2mL 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#780286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Air-permeable plate seal</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">60941-086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">96-pin plate replicator</td>
			<td style="width:108px;">Nunc</td>
			<td style="width:119px;">250520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacto-peptone</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">90000-368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacteriological agar</td>
			<td style="width:108px;">Affymetrix/USB</td>
			<td style="width:119px;">10906</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Ahringer</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">C. elegans RNAi Collection (Ahringer)</td>
			<td>See also Kamath et. al, Nature 2003.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Vidal</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">C. elegans ORF-RNAi Resource (Vidal)</td>
			<td>See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">WormLife- Software for Replica Set Survival Analysis</td>
			<td style="width:108px;">Samuelson Lab</td>
			<td style="width:119px;">N/A</td>
			<td>https://github.com/samuelsonlab-urmc/wormlife</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L4440 Empty Vector Plasmid</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">1654</td>
			<td>https://www.addgene.org/1654/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Wormbase</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.wormbase.org/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">OASIS</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://sbi.postech.ac.kr/oasis2/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Graphpad Prism</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://www.graphpad.com/scientific-software/prism/&#160;</td>
		</tr>
	</tbody>
</table>

the replica set method: a high-throughput approach to quantitatively measure caenorhabditis elegans lifespan

复制集方法是一种以高通量方式定量测量秀丽线虫的寿命或存活的方法, 从而使单个调查人员能够在相同数量的时间内筛查更多的治疗或条件。没有数据质量损失的时间。这种方法需要在大多数实验室中发现的常见的设备与C. 线虫, 因此是简单的采用。该方法的中心是在每个观察点上对一个种群的独立样本进行化验, 而不是随着时间的推移与传统的纵向方法进行抽样。评分需要在多井板块的井中加入液体, 这会刺激C 线虫移动并促进量化 healthspan 的变化。复制集方法的其他主要好处包括减少琼脂表面暴露于空气中的污染物 (例如霉菌或真菌), 对动物的最小处理, 以及对零星错误评分的鲁棒性 (例如, 当动物仍然活着)。为了对副本集样式实验中的数据进行适当的分析和可视化, 还开发了一种自定义软件工具。该软件的当前功能包括绘制副本集和传统 (卡普兰) 实验的生存曲线, 以及副本集的统计分析。这里提供的协议描述了传统的实验方法和副本集方法, 以及对相应数据分析的概述。

在任何新的方法的发展中, 新方法概括从以前的方法中接受的结果, 并符合领域内的标准势在必行。我们以前的经验表明, 复制集和传统的测定C. 线虫寿命的方法产生类似的结果20。野生型线虫(N2) 维持在20摄氏度通常生活在20和25天之间, 我们观察到的传统 (图 4A, 黑线) 和副本集方法 (图 4B, 黑色)。?...

Watch this Scientific Journal Video about The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan at JoVE.com

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

在这里, 我们描述了复制集方法, 一种定量测量C. 线虫寿命/生存和 healthspan 的方法, 以高通量和健壮的方式, 从而允许筛选许多条件而不牺牲数据质量。该协议详细介绍了策略, 并提供了用于分析副本集数据的软件工具。

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

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The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

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