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。

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作者声明他们没有竞争的财政利益。

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储存。这样, 在实验设置时, 每个应变的样本都被保留下来, 为将来的研究和提高重现性创造了宝贵的资源。然而, 虽然次氯酸钠治疗妊娠成年动物是一种常见的方式获得同步动物的衰老研究22, L1 逮捕是一个饥饿的反应。因此, 一些实验室倾向于允许孵化发生在盘子上, 或完全放弃次氯酸盐治疗, 并允许一些怀孕的成年人产卵几个小时 (即鸡蛋躺)。在后一种情况下, 父母被删除, 后代的寿命被遵循。据我们所知, 在 M9 的动物, 在盘子上孵化, 或从卵产卵的后代之间, 没有明显的寿命差异。然而, 鉴于营养可用性的变化与寿命的变化密切相关, 在这些同步方法之间, 特定的遗传背景可能产生不同的结果。为了解决这一理论问题, 需要进行更细致的分析。
无论用于同步起始种群的方法是什么, 都必须采取步骤以防止后代生产或将同步开始的人口与未来后代分离。在我们的协议中, 我们概述了如何使用 FUdR 来防止后代的产生, 因为分离动物不是复制集方法的可行选择。还可以通过使用女性化的遗传背景 (例如 15 (b26) 来防止后代生产, 而有限元-1 (hc17),这是一个温度依赖性的无菌菌株23)。然而, 两者都没有缺点: 遗传背景的使用会使随后的分析复杂化, 在某些遗传背景下 FUdR 可以改变长寿24,25,26。
作为防止后代通过化学或遗传手段生产的替代品, 成年动物可以周期性地移到新鲜的 rna 板上, 使其与后代隔绝。这就以牺牲吞吐量为代价简化了背景考虑。定期将动物迁移到新鲜食物有防止可能的饥饿和更新接触 dsRNA 的额外好处。然而, 一些影响寿命的基因相互作用只有在后代生产受到抑制时才发现: 生命表型的 TGFβ通路的早期分析错误地断定, TGFβ信号的减少影响了线虫dauer 形成但不老化27,28。然而, 使用 FUdR 的后续研究显示, TGFβ信号的减少, 通过胰岛素信号29增加了长寿。为什么早期的研究不能看到 TGFβ变种动物的寿命增加？TGFβ路径突变产生轻微的产卵缺陷 (egl) 和延长繁殖寿命, 这导致后代的内部孵化后, 生命中杀死的父母。很可能长寿的 TGFβ变种动物似乎有正常的寿命, 因为egl表型杀死动物的时候, 野生型动物通常死亡。这可能与其他与 DR 相关的基因通路有关, 因为饥饿的野生型动物也表现出一种egl表型, 也许是在低食物条件下后代的适应性生存优势。这突出了动物在压力条件下的适应性反应的基本复杂性, 以及在设计寿命试验时需要仔细分析和考虑的问题。
在设计和进行寿命试验的任何一种方法, 这是至关重要的, 以避免偏差。实验必须以双盲的方式进行: 以前的时间点上的样本是如何得分的, 测试条件的身份必须是实验者所不知道的。此外, 始终必须包括正反两方面的控制;在复制集方法的情况下, 这些被随机地插入到一个24井板中。大肠杆菌表达的质粒不含有与C. 线虫基因组对应的序列的插入物, 是空矢量负控制 (即"L4440"-见材料)。积极的控制取决于实验的具体性质。例如, daf-2编码的C. 线虫insulin/IGF-1 受体, 和daf-2失活通过喂养基的干扰有力地增加寿命至少两倍的野生型动物27。因此, 2 (rna 干扰)可能作为一个积极的控制, 当寻找基因 inactivations, 增加寿命。反之, daf-16编码 foxo 蛋白转录因子 orthologue30。DAF-16 是许多长寿范式的重要组成部分, 野生型动物 (N2) 处理的16 (rna)是短命的和显示的迹象, 早衰31。
传统的纵向寿命方法的主要优点是它建立得很好, 实验容易建立。在每个试验条件下只需要几个盘子就能得到相对较少的动物。因此, 生长不良或需要平衡器传播的菌株可以很容易地被测试。传统的方法具有很强的适应性, 可用于处理后代生产的任何一种现有方法, 包括 FUdR 治疗、杂交成女性化的遗传背景, 或定期将成年动物移到新板块在产卵期间。虽然移动的动物大大降低了吞吐量, 但使用突变体的背景从来都不是理想的, 虽然 FUdR 不改变野生型寿命32,33,34, 它可以影响寿命和年龄相关的表单在一些遗传背景35,25,26。注意, 雄性的存在, 即使使用 FUdR, 也会显著缩短13的雌雄同体寿命, 因此在雌雄同体到达 L4 后, 含有雄性的盘子是不可用的。同样, 通过卡普兰-梅尔估计和相关曲线的分析, 以及对数秩测试, 对死亡率数据也有很好的确定。然而, 传统的寿命分析存在着一些弊端。反复处理板材 (即将盘子暴露在空气中) 有助于引入空气中的真菌污染。此外, 反复戳可以伤害或杀死动物, 特别是随着人口的老龄化和变得脆弱。年长的动物基本上瘫痪并陷入大肠杆菌,而大肠杆菌则成为一种机会主义病原体 (殖民腔和包装咽)36。非常古老的活体动物只能通过微妙的头部运动来识别。因此, 将一只衰老的活动物归类为死亡是很容易的。最后, 传统的方法受到吞吐量的限制。
副本集方法具有较高的吞吐量和定量。但是, 这种方法的缺点是在初始设置中投入大量的时间和资源。一个适度大的实验, 以检查100个 rna 变异克隆超过20个时间点需要 3万 L1 动物 (其中大约15个动物每次 rna 干扰克隆检查), 虽然容易对多数菌株, 在某些情况下可能是疑难的。例如, 如果没有一个大粒子分类器 ("蠕虫分拣") 菌株, 必须保持与平衡器或转基因线携带一个不良的外染色体阵列不能很容易地检查此方法。第二个缺点是必须抑制后代的生产, 这需要使用 FUdR 或女性化的遗传背景。最后, 你必须知道化验将运行的时间长短, 因为你必须准备一个副本集为每个时间点在实验开始。然而, 这种方法的优点很多。最重要的是, 得分的可行性要快得多, 你可以很容易地跟踪动物100多个测试条件同时 (即rna 克隆)。由于复制品集只被打一次然后丢弃, 没有重复处理的板块或戳动物, 这将减少真菌污染的可能性, 并消除由偶尔粗暴刺激的蠕虫采摘造成的死亡率。此外, 液体在井中的加入大大促进了评分。从盘子和周围的细菌中释放老动物有助于使细微的头部运动更容易得分。添加液体也提供了一个机会, 以测量颠簸率作为衡量的健身 (如healthspan8,37)。
衰老是一个复杂的现象, 涉及多种因果机制, 需要使用系统的生物方法来解开。这些方法通常结合数据驱动的建模, 使用大量的基因组/transcriptomic 数据, 并需要互补的健壮和高通量的方法来测量寿命和 healthspan。高通量复制集方法可以纵向比较许多 rna 干扰克隆, 同时尽量减少批处理效果和技术误差, 从而促进动态模型的开发, 从而可以推断因果通路之间的相互作用。定量的方式。此外, 由于大量年龄同步的动物被划分成少数群体, 因此, 将几个全基因组学方法与复制集方法结合起来是可行的。
以前已经开发了其他方法来提高C. 线虫寿命试验的吞吐量, 通常侧重于适应传统的纵向方法 (即跟随同一组动物随着时间的推移), 以自动化观察和记录使用普通平板扫描仪38,39, 或更专业的设备, 如微流控板40。基于扫描仪的方法使用光作为刺激, 并比较按顺序捕获的图像, 以确定活/死的状态, 基于移动多个板块的一次;虽然这种方法不需要专有的科学硬件, 但根据所需的规模, 设置工作流所涉及的时间可能很大。另外, 自定义微流控装置的寿命试验允许在一段时间内对单个动物进行深入的表型表征, 而无需治疗以防止后代, 但需要制作微流控板和获取相关的微流控泵和成像设备。与此相反, 复制集方法与此处详细介绍的软件相结合, 可以极大地提高吞吐量, 使用在与C. 线虫一起工作的实验室中已经常见的工具。
WormLife 软件将在将来得到改进, 以便更方便地访问统计比较, 并与其他平台兼容。该软件的最新文档可以在 GitHub 页面找到, 包括测试软件的平台的安装说明。还将开发一个基于 web 的版本, 以便在不需要安装任何软件的情况下方便地访问。
总之, 复制集方法和可用的免费软件的组合提供了一个强大的平台, 用于提高寿命实验的吞吐量和健壮性, 以及广泛的基于生存的测试 (例如,应激耐受、毒理学研究、healthspan等)。特别是当与功能基因组学结合时, 这种方法利用了后生模型系统C. 线虫的许多好处来破译无数的基因相互作用, 从而促进衰老的进程.

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)。因此, 这两种方法都合理地近似野生型寿命。同样重要的是, 一个新的方法有决心准确地量化测试条件和统计能力之间的变化, 以检测重大变化。在我们之前的研究中, 我们发现, 转录因子的 c-myc 家族是C. 线虫长寿的决定因素。mml-1和mxl-2编码的同系物哺乳动物蒙多 A/碳水化合物反应元素结合蛋白 (ChREBP) 和 Mlx 分别。在线虫和哺乳动物中, 这些 c-myc 家族成员 heterodimerize 调节转录。我们发现, 无论是mml-1还是mxl-2的丢失都显著降低了正常的寿命, 这是通过传统的寿命分析或复制集 (图 4A-B, 黄色和栗色) 来衡量的。与 MML-1::MXL-2 复杂, 我们发现, mdl-1 (同源的哺乳动物疯狂) 或mxl-1 (最大) 的损失显著增加了 C. 线虫的寿命, 用两种方法来衡量 (图 4紫色和蓝色, 分别在两个面板)。
对传统的寿命测量方法的严重限制是吞吐量。这两种方法都依赖于运动来调用动物是活的还是死的, 这变得越来越难以评估。幼兽在没有刺激的情况下会在盘子里移动, 因此很容易得分。老化的线虫变得越来越久坐, 但将响应一个轻触头由反转运动的板块。然而, 随着动物变老, 向后移动的能力减弱, 变得越来越不协调。最终动物变得瘫痪, 一个表型强烈类似于骨骼肌减少, 当通过传统的方法进行评分时, 只能通过观察动物极端前尖端的细微抽搐来确定。相比之下, 当通过复制集方法得分时, 液体被添加到井中, 这是一种刺激, 它产生了一个可量化为 healthspan8读数的抖动响应。液体中的运动更容易观察到更老的动物: 按年代年龄配对的衰老动物在干板上产生微妙的头部运动, 但在液体中更明显 (虽然缓慢) 身体弯曲。最后, 当评分副本集, 整个井是在视野 (1.1 厘米直径) 和所有动物都在悬浮-允许观察所有动物同时。相比之下, 当通过传统的方法评分6厘米板, 你必须扫描整个板块搜索通过细菌草坪和沿边缘为动物。这些差异的净结果是, 使用副本集方法时的吞吐量至少比传统方法大一级, 这使得可以同时量化超过100的寿命变化。条件在一个实验与一个调查员。例如, 从一个全基因组的基于喂食的 rna 干扰屏幕, 我们以前确定了159基因是必要的增加寿命, 由减少daf-2/胰岛素样信号8。在这一分析中, 我们量化了野生型的寿命变化, 一个长期存在的2 (e1370)突变体, 和短命的 e1370 (2);d af-16 (mgDf47)双突变动物 (图 5A), 这使我们能够破译遗传胰岛素样信号与 100 progeric 基因 inactivations 的关系。此外, 我们评估了这些 progeric 基因 inactivations 如何改变 healthspan (当时称为 "activespan") 通过观察的下降,线虫跨越复制一段时间 (图 5B)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57819/57819fig1.jpg"> 图 1: 基于喂食的 rna 干扰的出现导致了衰老研究中基因发现的时代, 然而大多数 gerogenes 仍然缺乏研究.(A)许多 gerogenes 最初是从大规模功能基因组筛中发现的。在迄今发现的900多种线虫gerogenes 中, 许多都是使用基于喂养的 rna 识别技术, 突出了功能基因组方法在基因发现中的价值。该图显示了每篇手稿所发现的 gerogenes 的数量, 其方法是基于表型标注 (见材料表), 用于表型本体术语延长寿命, 缩短寿命和寿命变体。有关发现 gerogenes 的完整研究列表, 请参阅补充文件 1 。(B)大多数 gerogenes 仍然缺乏研究。与研究 gerogenes 类似的daf-16/foxo 蛋白 (箭头) 相比, 它有800多个参考文献, 但大多数 gerogenes 的参考文献少于10个 (一般参考, 不一定侧重于寿命)。可靠的高通量方法对于深入了解 gerogenes 之间的遗传相互关系是至关重要的。该图基于 PubMed 中的出版物与 gerogenes 的 rna 表型中发现的C. 线虫之间的映射。有关 gerogenes 的完整列表和与之相关的研究数量, 请参阅补充文件 2 。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57819/57819fig2.jpg"> 图 2: 传统的和副本集方法, 用于评分C. 线虫寿命 (A)。对线虫寿命的传统评分方法。随着时间的推移, 等基因系动物的几个小的同步种群。在整个学习过程中都遵循同样的动物种群。活力是通过运动来评估的, 这可能受到温和的刺激。不能移动的动物被评分为死, 并被删除 (显示的愿望), 直到没有活着的动物留下。(B).用于评分C. 线虫寿命的复制集方法。大量的年龄同步的等基因系动物分布在多个相同的复制板上。在每个时间点, 一个单一的复制被评分: 一个温和的缓冲解决方案 (M9) 增加, 刺激运动。在注水井后不能自发移动的动物也通过触控刺激来评估。实验的评分持续时间是在开始之前确定的。每只动物只得分一次, 更大的人口的长寿来自许多独立观察。(c).复制集方法是一种用于定量测量线虫寿命的高通量方法。可以同时跟踪100个或更多独立的 rna 干扰克隆。HT115大肠杆菌表达 dsRNA 的一个特定的 rna 干扰克隆显示。实际上, 从96井板中抽取的每24个样品都被分成一个24井板。每一个产生的24井板有一个负 (即空矢量, 红色井) 和积极控制 (绿色井) 随机分布在一个收集的 rna 干扰克隆 (黄色水井)。通常, 集合中的第一个井 (A1) 包含一个空向量。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57819/57819fig3.jpg"> 图 3: 图形用户界面 (GUI)。(A).显示默认欢迎屏幕的主绘图窗口界面。这是打开软件时显示的内容。由于使用平台无关的窗口化工具包, 平台之间的外观差异应该是最小的。(B).菜单选项概述, 用于从主绘图窗口提供的下拉菜单。(C).复制副本集样式 (左) 和传统卡普兰-Meier 样式 (右) 数据的绘图输出示例。所显示的数据是在独立实验中收集的。导出的地块不包括预先绘制的坐标轴标签, 以便在添加此类标签时具有最大的灵活性。为便于实现此目的, 坐标轴总是被划分为 Y 轴的20% 增量, X 轴的增量为5。在此示例中, 使用非常简单和常见的图像编辑工具, 将坐标轴和线条标签 (应变/处理) 添加到保存的图中。(D).从 "TrialView" 功能输出的一个示例, 允许对副本集样式数据集的独立试验结果进行可视比较。这个情节显示了两个不同的试验结果和相应的汇集结果为daf-2 EV (rna) (蓝色, 闭合的圈子), N2 EV (rna) (黑, 闭合的圈子) 和daf-2与daf-16 (rna 干扰) (红色, 开放金刚石)。TrialView 允许快速检查可能影响池数据集匹配质量的特定于试验的数据问题。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57819/57819fig4.jpg"> 图 4: 传统和副本集方法产生相似的结果.1 (Mad) 的任一组分的损失:: MXL-1 (最大) heterodimer 增加寿命。相比之下, MML-1 (蒙多/ChREBP) 的任一组成部分的损失:: MXL-2 (Mlx) 降低寿命这一数字是从参考20重印, 通过一个创造性的共同性归属 (CC) 许可证 (见材料)。(A).卡普兰-梅尔结果与传统方法。(B). Logit 曲线适合使用副本集方法。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57819/57819fig5.jpg"> 图 5:复制集方法可以根据寿命 (A) 的变化和 healthspan (B) 中100个 rna 克隆同时进行的改变来破译遗传相互作用。这个数字被重印从8在创作共同性归因于的允许之下-非商业4.0 国际执照 (由 NC) (参见材料)。(a) progeric 基因 inactivations 的遗传寿命分析在胰岛素样信号 (ILS) 减少 (daf-2, x 轴) 和缺乏daf-16/foxo 蛋白 (Y 轴) 的情况下, ILS 21 的中枢转录效应器.基因 inactivations 具有类似的功能, daf-16在没有daf-16 (黑点) 的情况下不会进一步缩短寿命。基因 inactivations 与功能完全地独立从daf-16缩短两个基因背景相似地 (灰色)。基因 inactivations 在daf-2 > daf-2;daf-16 中对生命周期产生负面影响,提示平行 (白色) 功能。(B)随着时间推移, 颠簸率的变化可以得出许多遗传扰动 (x 轴) 的平均 healthspan, 同时评估寿命的变化 (y 轴)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/57819fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
图 S1: WormLife 中的工作流.一些引导工作流 (有时称为 "向导") 步骤的说明。在这些情况下, 在工作流的最后一步之后, 焦点将返回到主绘图窗口。(A)复制副本集样式集的数据导入工作流(B)传统的卡普兰 Meier 样式数据集的数据传输工作流。(C)将行添加到副本集样式数据集的绘图中的工作流。(D)为传统的卡普兰 Meier 样式数据集添加线条到绘图区的工作流。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/figure_S1.tiff">请单击此处下载此文件.</a>
补充文件 1: 已确定 gerogenes 的研究.基于喂食的 rna 干扰的出现导致了一个基因发现的时代, 它不是由前遗传学 (包括衰老) 驯服的。按发现的 gerogenes 数量顺序列出的是独立的研究, 它确定了其活动改变了寿命的基因。请注意, 识别最 gerogenes 的研究使用了复制集方法8。基因 inactivations 改变寿命的性质也表明: 长寿和 progeric 基因是那些增加或降低生命周期时, 分别。"寿命变体" 指的是没有指定或未被策划的变化方向性 (即增加或减少寿命) 的情况。来自 WormBase WS262 (2018年1月) 的数据 (见材料), 加上参考文献10的 rna 处理寿命结果, 这还未包括在精心策划的 WormBase rna 表型的收集中。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_1.xlsx">请单击此处下载此文件.</a>
补充文件 2: 与每个 gerogene 相关的研究的数量.大多数 gerogenes 研究得不好。虽然一些基因, 如daf-16/foxo 蛋白, 一直是研究关注的主题, 超过 400 gerogenes 有少于10相关的出版物。数据从 WormBase WS262 (2018年1月), 加上干扰治疗寿命的结果从10 , 其中尚未包括在精心策划的收集 WormBase rna 干扰表型。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_2.xlsx">请单击此处下载此文件.</a>
补充文件 3: 制备常用试剂C. 线虫实验。(A)标准 NGM 和 rna 干扰板的配方。(B) M9 缓冲液和次氯酸盐溶液的配方。(C)制备 LB/春节版。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_3.xlsx">请单击此处下载此文件.</a>
补充文件 4: 副本集示例数据.副本集寿命试验中的一个示例数据集。此数据集已格式化为适合导入/分析。包括每一个条件的两个试验 (应变/基因型和 rna 干扰的组合)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_4.csv">请单击此处下载此文件.</a>
补充文件 5: 传统的纵向示例数据.一个例子数据集从传统的纵向寿命实验, 与右审查, 格式化的准备进口/分析使用卡普兰-梅尔生存情节功能。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57819/Supplemental_File_5.csv">请单击此处下载此文件.</a>

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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Nanyang Technological University

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JoVE Journal

Genetics

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

8.5K 次观看.  University of Rochester Medical Center. 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。

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

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

科研

遗传学

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

大规模混合阶段 卡诺哈布迪蒂斯电子 人种群的文化和分析

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

糖精中遗传链接调控时间寿命的抑制 器屏幕

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...