Name: using caenorhabditis elegans for studying trans- and multi-generational effects of toxicants
Uploaded: 2019-07-29T12:30:00
Description: Watch this Scientific Journal Video about Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants at JoVE.com

1. 培养大肠杆菌OP50
<ol><li>通过在100 mL水中溶解4克氢氧化钠溶液制备1M氢氧化钠溶液。</li>
	<li>在1L锥形烧瓶中溶解10克试金石、5克酵母提取物和10克氯化钠,制备溶质汤(LB)培养基。使用 1 M 氢氧化钠溶液将 pH 调整到 7.0。</li>
	<li>将 LB 液体介质从步骤1.2压成 20 锥形烧瓶(最大允许体积:100 mL),每个介质为 50 mL 介质。用牛皮纸盖住圆锥形烧瓶。</li>
	<li>在 121°C 和 0.105 MPa 下对 LB 液体介质进行消毒...

<ol>
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为了成功实施所述协议,应考虑以下建议。在无菌环境中执行总体实验操作。操作不当可能导致大肠杆菌菌株(如真菌和虫子)的污染,从而阻碍大肠杆菌的正常生长,从而影响实验结果。在描述培养C. elegan 的章节中,用肉眼或显微镜观察 NGM 琼脂上C. elegans的生长尺度。当琼脂的C.elegans在面积上超过75%,或养殖时间超过一周时,进行新一轮的接种,以避免C.elegans的过度...

化学品的应用和废物管理高度依赖于其在特定浓度下的影响信息。值得注意的是,时间是影响和浓度之间的另一个基本要素。也就是说,化学品,特别是那些在实际环境中浓度低的化学品,需要时间来产生可衡量的效果。因此,研究人员在动物实验中安排了不同长度的暴露时间,甚至覆盖了整个生命周期。例如,老鼠接触尼古丁30天,90天或180天,以研究其毒性影响2。 然而,这种接触持续时间仍然不足以阐明污染物(例如持久性有机污染物[持久性有机污染物])的长期影响,这些污染物在环境中可以持续几代生物。因此,对几代人影响的研究越来越受到人们的重视。
代际效应研究主要有两个方面。第一个是跨代(TG)效应研究,它可以有力地测试与父母接触的化学物质是否会对后代造成任何后果3。第二个是多代效应研究,在接触和残余效应方面考虑更系统。一方面,多代暴露(MGE)效应用于说明动物对长期具有挑战性的环境的适应性反应。另一方面,多代残留效应用于证明接触后的长期残留后果,因为母体接触伴随着胚胎接触第一个后代和胚芽系接触第二个后代后代,使第三代作为第一代完全出暴露4。
虽然哺乳动物(例如,小鼠)是毒性研究中的模范生物体,特别是与人类有关的生物体,但在研究代际影响时,它们的应用相当耗时、昂贵和伦理上涉及5。 因此,包括甲壳类达芙尼亚麦格纳6号、昆虫果蝇黑色素7和斑马鱼达尼奥雷里奥8号在内的生物提供了替代选择。然而,这些生物要么缺乏与人类的相似性,要么在研究中需要特定的设备。
Caenorhabditis elegans是一种小型自由活线虫(长约 1 毫米),生命周期短(在 20 °C 时约为 84 小时)9。这种线虫与人类有许多保守的生物途径,因此被广泛用于说明各种应力或毒物的影响。值得注意的是,99.5%的线虫是甲虫,使这种生物极适合研究代际效应,例如重金属和磺胺的TG效应3,11,金纳米粒子和重重的MGE效应金属12和温度13,MGR效应的磺胺14,以及MGE和MGR效应的伽马辐照15和林丹4。此外,在化学品(如Zearalenone)对小鼠发育和繁殖的影响和16、17的C.elegans之间发现了类似的结果,这将为推断从这个小动物到人类的影响。
TG 和 MG 效应研究既耗时,也需要仔细的设计和性能。值得注意的是,上述研究中在生命阶段选择、暴露条件和代际分离方法方面存在差异。这种差异妨碍了结果之间的直接比较,也妨碍了对结果的进一步解释。因此,必须建立统一的协议,以指导TG和MG效应研究,并提供一个更大的图景,揭示各种有毒物质或污染物在长期后果中的相似模式。现行协议的首要目标将表明在研究C.elegans的跨代和多代效应方面的明确操作过程。这些协议将有利于那些有兴趣研究有毒物质或污染物的长期影响的研究人员。

<table border="0" cellpadding="0" cellspacing="0" style="width:684px;" width="684">
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			<td height="37" style="height:37px;width:191px;">&#160;agar powder</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td style="width:123px;">9002-18-0</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">79nnHT Fast Real-Time PCR System</td>
			<td>&#160;Applied Biosystems&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">96-well sterile microplate</td>
			<td style="width:200px;">Costar&#65292;Corning&#65292;America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Autoclave sterilizer</td>
			<td style="width:200px;">Tomy, Tomy Digital Biology, Japan</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Biosafety cabinet</td>
			<td style="width:200px;">LongYue, Shanghai longyue instrument equipment co. Ltd, China</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">calcium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">centrifuge&#160; 5417R</td>
			<td style="width:200px;">Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Centrifuge tubes</td>
			<td style="width:200px;">Axygen, Aixjin biotechnology (Hangzhou) co. Ltd, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">cholesterol</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">57-88-5</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Dimethyl sulfoxide</td>
			<td style="width:200px;">VETEC, Sigmar aldrich (Shanghai) trading co. Ltd, America</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">disodium hydrogen phosphate</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">7558-79-4</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">ethanol</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td style="width:123px;">64-17-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Filter</td>
			<td style="width:200px;">Thermo, Thermo Fisher Scientific, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">incubator</td>
			<td style="width:200px;">YiHeng17, Shanghai yiheng scientific instrument co. Ltd, China</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">inoculating loop</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">K2HPO4&#8226;3H2O</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>16788-57-1</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">kraft paper</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Mcroplate Reader</td>
			<td style="width:200px;">Boitek, Boten apparatus co. Ltd, America</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MgSO4&#8226;7H2O</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Microscopes XTL-BM-9TD</td>
			<td style="width:200px;">BM, Shanghai BM optical instruments manufacturing co. Ltd, China&#160;</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Petri dishes</td>
			<td style="width:200px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">Pipette</td>
			<td style="width:200px;">Eppendorf, Ai Bende (Shanghai) International Trade Co., Ltd, Germany</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Potassium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">potassium dihydrogen phosphate</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7778-77-0</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Qiagen RNeasy kits</td>
			<td style="width:200px;">Qiagen Inc., Valencia, CA, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">QuantiTect SYBR Green RT-PCR kits</td>
			<td style="width:200px;">Qiagen Inc., Valencia, CA, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:191px;">RevertAid First Strand cDNA Synthesis Kit</td>
			<td style="width:200px;">Thermo Scientific, Wilmington, DE, United States</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">sodium chloride</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">sodium hydroxide</td>
			<td style="width:200px;">Sinopharm chemical reagent company Ltd, China</td>
			<td>1310-73-2</td>
			<td></td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;">sodium hypochlorite solution</td>
			<td style="width:200px;">Aladdin, Shanghai Aladdin biochemical technology co. Ltd, China</td>
			<td style="width:123px;">7681-52-9</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">tryptone</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td>73049-73-7</td>
			<td style="width:171px;"></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">yeast extract</td>
			<td style="width:200px;">OXOID, Thermo Fisher Scientific, UK</td>
			<td>119-44-8</td>
			<td style="width:171px;"></td>
		</tr>
	</tbody>
</table>

using caenorhabditis elegans for studying trans- and multi-generational effects of toxicants

有关化学品毒性的信息对于化学品的应用和废物管理至关重要。对于低浓度化学品,长期影响对于判断其对环境和人类健康的影响非常重要。在展示长期影响时,化学品对几代人的影响为最近的研究提供了新的见解。在这里,我们描述了使用自由生活线虫Caenorhabdiselegans研究多代化学物质影响的协议。提出了两个方面:(1)跨代(TG)和(2)多代效应研究,后者分为多代暴露(MGR)和多代残差(MGR)效应研究。TG效应研究是强大的,其目的很简单,即与父母接触化学品是否会对后代造成任何残留后果。在测量对父母的影响后,使用次氯酸钠溶液杀死父母并保留后代,以便促进对后代的影响测量。TG效应研究用于确定当后代的父母暴露于污染物时是否受到影响。MGE 和 MGR 效应研究用于系统化,用于确定连续的代接触是否会导致后代代代适应反应。小心的拾取和转移用于区分代,以方便对每一代人进行效果测量。我们还结合协议来测量运动行为、生殖、寿命、生化和基因表达变化。还给出了一些实例实验来说明跨代和多代效应研究。

在这里,我们描述了在跨代(TG)、多代接触(MGE)和多代残留(MGR)效应研究中使用C.elegans研究化学物质对几代人的影响的协议。我们自己的研究结果作为例子提出。一项研究介绍了重金属对运动行为3的TG影响。其他两项研究显示硫磺胺和林丹对繁殖和生化及遗传指数测量4、14的MGE和MGR效应。
<p class="jove_conten...

Watch this Scientific Journal Video about Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants at JoVE.com

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

使用卡内哈布迪炎酶研究有毒物质的跨代和多代效应

持久性化学品的跨代和多代影响对于判断其对环境和人类健康的长期影响至关重要。我们提供了研究跨代和多代效应使用自由生活线虫卡埃诺哈布迪炎埃莱甘斯的新方法。

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

提出的方案将促进对蛋白质的跨代效应和多代效应的研究，这些蛋白质可以在环境中长期存在。我们使用了一种具有 99.5 的免费线虫作为草本虫，以节省研究跨代和多代效应的时间和精力。这种技术可以用来证明最终药物的长期影响，因为本协议中的线虫与人类有许多保守的机制或通路。这种方法可以提供对药物合成和环境管理领域的见解，因为它们需要对化学品的潜在危害的反馈。该方法可应用于达芙尼亚磁石，更注重水生毒性。此外， 德罗斯菲拉黑色素， 以进一步探索对性别的影响。他或她会坚持这样的想法，即研究几代人的影响是耗费时间和精力的。一开始不要害怕。遵循协议将赢得您丰硕的成果。为了培养大肠杆菌，移液器200微升从细菌悬浮液到50毫升的LB介质在烧瓶。或者使用接种回路从NGM agars中挑选一个小菌群，并放在LB介质中。为了培养C.elegans，在显微镜下，数一数之前准备的堆叠NGM阿加的线虫。当数字接近2000时，使用无菌尖端切割1/6的NGM阿加，并转移到新准备的NGM阿加与细菌草坪。将 NGM 阿加在 22 摄氏度的培养箱中倒置，以进行后续培养。大约36小时后，线虫变得格格不长。使用两毫升无菌水将每个NGM阿加上刚生产的麦线虫和新生产的鸡蛋冲洗到无菌离心管中。将管子放在架子上 30 分钟，以安定线虫，然后通过移液丢弃 85% 的超自然剂。判断线虫是否足够高，可以同步是非常重要的。否则，年龄同步操作将徒劳无功。将颗粒与七卷次氯酸钠溶液混合，每两分钟摇动离心管五至八次，以解液分离幼虫和成年线虫。在20摄氏度下以700倍G离心管3分钟，然后通过移液将超级钠丢弃。将颗粒重新悬浮在五卷无菌水中，以清洗年龄同步的鸡蛋。在 20 摄氏度下以 700 倍 G 离心三分钟。用无菌水重复洗涤两次。接下来，在管子中加入一卷无菌水，重新悬浮与年龄同步的鸡蛋。通过移液 50 微升将鸡蛋悬浮液分发到每个新的 NGM 阿加子上，并配有细菌草坪。将NGM阿加在20摄氏度的培养箱中保持顶部，30分钟，让水蒸发或被细菌草坪吸收。然后，翻转NGM阿加，倒置，为随后的文化，并标记时间作为鸡蛋时间。卵子时间36小时后，线虫到达L3幼虫阶段。使用两毫升无菌水将线虫从每个NGM气量中冲洗到离心管中。沉淀30分钟后，用K中等液替换85%的超级钠，消化肠道中的食物两小时。然后，丢弃超级纳特。使用 K 中等将线虫悬浮液调整为每 100 微升约 200 线虫，以便进行后续实验。为了避免边缘效应，在96孔无菌微孔板的中间区域，将100微升的准备好的对照或化学溶液加入10孔，如6组复制。在60口井中，每口加入100微升的准备好线虫K介质。将时间标记为 T0。执行曝光 24 至 96 小时。曝光后，使用移液器从每组5个孔中收集线虫，进入6个1.5毫升离心管。将线虫沉淀30分钟，然后移液器丢弃超钠。用一毫升消毒水清洗底部的线虫。将线虫沉淀30分钟。丢弃超超自然剂，并使用底部的线虫进行暴露父代的指示器测量，标记为 F0。从每组剩余的五口井中收集线虫。如前所述，沉淀和清洗线虫。接下来，加入100微升无菌水，重新悬浮每个管中的线虫，并将线虫悬浮液均匀地转移到三个新准备的NGM加糖上，用细菌草坪。孵育线虫36小时，成为格拉维德，并执行年龄同步，如前所述。在NGM agars上用细菌草坪孵育同步的卵子36小时后，将线虫冲洗成6个离心管。使用一毫升灭菌水进行沉降和洗涤后，使用底部的线虫对标记为 T1 的后代进行指示器测量。将 99 毫升温暖的 NGM agar 与一毫升的控制或化学溶液混合。根据手稿，在用细菌悬浮物准备阿加斯后，在生物安全柜中，将培养皿的上盖放在一边，将细菌草坪暴露在紫外线下 15 分钟。将年龄同步的鸡蛋移到经过紫外线处理的糖上。将父代 F0 的曝光的开始标记为天零。在20摄氏度下孵育三天。然后，使用成熟的线虫测量F0的效果。此外，第三天，使用装有人造纤维线弯曲成环的玻璃棒来挑选F0成熟的线虫，然后滑到新的经过紫外线处理的NGM阿加的表面上。第四天，从NGM agars中挑选并丢弃成熟的F0线虫。第六天，使用经过三天暴露的F1成熟线虫进行指数测量。在该协议中，镉、铜、铅和锌对身体弯曲频率的跨代效应表明，产前接触后线虫亲亲F0的抑制作用小于其后代T1。磺胺多代残留对线虫寿命和生殖的影响表明，生殖在病菌线暴露中受到显著影响，毒性在T3代至T6代无暴露的代代中持续存在。林丹对线虫生化和遗传指数的多代接触和多代残留效应表明，胰岛素信号调节中的干扰作用有致畸作用。此外，sgk-1和 akt-1 信号之间的变化表明，不同接触代的线虫表现出不同的耐受和避免反应策略。在每次操作前检查线虫的生活状态，并相应地修改以下步骤。其他影响，例如，化学品的肥胖效应，可以包括进一步回答肥胖在几代人中日益普遍的问题。在此基础上，可以探索出进一步的机制。例如，世代之间的遗传表观遗传信号。氯溶液具有很强的氧化能，避免皮肤直接接触。

Research

JoVE Journal

Environment

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

技术树内组织学习节肢动物和微生物群落

Phloem tissues of pine are habitats for many thousands of organisms.  Arthropods and microbes use phloem and cambium tissues to seek mates, lay eggs, rear ...

科研

环境科学

Measuring Fluxes of Mineral Nutrients and Toxicants in Plants with Radioactive Tracers

植物与放射性示踪剂测量矿质营养和毒物的通量

Unidirectional influx and efflux of nutrients and toxicants, and their resultant net fluxes, are central to the nutrition and toxicology of plants. ...

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

A CO2 Concentration Gradient Facility for Testing CO2 Enrichment and Soil Effects on Grassland Ecosystem Function

一个外线<sub&gt; 2</sub&gt;浓度梯度用于测试CO<sub&gt; 2</sub&gt;浓度与土壤对草地生态系统功能的影响

Continuing increases in atmospheric carbon dioxide concentrations (CA) mandate techniques for examining impacts on terrestrial ecosystems. Most ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

A Flow-through Exposure System for Evaluating Suspended Sediments Effects on Aquatic Life

对水生生命值评价悬浮泥沙影响的流通系统曝光

This paper describes the Fish Larvae and Egg Exposure System (FLEES). The flow-through exposure system is used to investigate the effects of suspended ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

评估环境和遗传因素对鹿茸和长寿命的鹿的身体生长的相对影响的议定书 》

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

研究矿物对有机热液转化影响的实验协议

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

In Vitro Scratch Assay to Demonstrate Effects of Arsenic on Skin Cell Migration

在体外显示砷对皮肤细胞迁移影响的划痕测定

Understanding the physiologic mechanisms of wound healing has been the focus of ongoing research for many years. This research directly translates into ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

一种用自动表型分析预测线虫化学毒性的高通量测定

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...