The authors would like to thank the following funding sources NIH ES020353 and the Colgate-Palmolive Alternative Research Grant award for making this work possible.

1.准备喂食细菌注意：本节介绍喂食细菌（ 大肠杆菌菌株OP50）的准备。 <ol><li>隔离大肠杆菌的单个菌落从LB培养基（LB）琼脂平板上，并在无菌条件下大肠杆菌菌株OP50接种入300ml高压灭菌的LB肉汤。 </li><li>允许接种培养过夜在摇床在200rpm和37℃下生长，直到达到饱和。 </li><li>转移OP50成6无菌，预先称重的50ml锥形管中。通过在6000转（5000 XG）离心5分钟沉淀细菌。 </li><li>弃去上清液，并用50ml无菌的M9洗细菌。重复两次。 </li><li>第二次洗涤后除去残留的M9，确保没有M9被留在管中。 </li><li>通过称量含有细菌沉淀管确定粒料的重量，并减去在50ml锥形管中的重量。 </li><li>悬浮颗粒我Ñ​​M9以100mg / ml的浓度。 </li><li>储存在4℃的OP50溶液，直到它被用于蠕虫培养。 </li></ol> 2.制备线虫生长培养基（NGM）培养皿注意：这部分描述NGM培养皿的制备，这是所述板，其中所述C。线虫蠕虫例行维持在实验室。 <ol><li>高压灭菌的NGM琼脂培养基。 </li><li>使用无菌的程序，免除17毫升的NGM琼脂溶液倒入用蠕动泵6厘米培养皿。 注：琼脂中平板恒定量减少了需要从一个板切换到另一个时再聚焦显微镜。 </li><li>离开所述板在室温下放置2-3天使用前，让琼脂固化，允许多余的水分蒸发。 </li><li>种子用无菌技术的NGM板。应用1-2滴OP50液体培养物并扩散用玻璃棒。确保不一路蔓延细菌草坪到板的边缘。 </li><li>允许OP50草坪在室温下用板成长蠕虫之前生长1-2天。 </li></ol> 3.准备一个同步蠕虫人口注：C。的生命周期线虫是由萌芽阶段，四幼虫阶段（L1-L4）和成年。本节描述了蠕虫的时代同步人口的制备。所有的材料来在漂白处理后与线虫接触必须是无菌的。 <ol><li> 第1天-妊娠蠕虫人口的制备 <ol><li>使用NGM板中的大多数蠕虫人口由饿死L1幼虫。 </li><li>收集L1幼虫用消毒虫挑，并将它们转移到新鲜6厘米NGM板接种OP50。孵育约65小时，在20℃，直到大部分的蠕虫是妊娠成人。到避免板拥挤并允许蠕虫未耗尽的细菌生长，不L1幼虫多于一个或两个簇转移到每个新鲜NGM板。 </li></ol></li><li> 4日-建立同步L1幼虫人口 <ol><li>通过与M9 1-2毫升冲洗它们收集来自NGM板的妊娠蠕虫和它们转移到一个锥形15ml试管。 </li><li>让妊娠蠕虫沉淀到15毫升锥形管的底部为约5-10分钟。去除上清，而不会干扰病毒颗粒。 </li><li>该蠕虫病毒颗粒转移到2-4离心管，并添加了漂白剂/氢氧化钠溶液1ml。孵育在室温2-3分钟。监视立体显微镜下的反应的进行，以确认所有的蠕虫都死了。不要漂白超过5分钟，因为胚胎会死亡。 </li><li>通过在3000转（900 XG）离心1分钟收集蠕虫。除去上清液，并添加无菌的M9 1ml至中和反应。 </li><li>通过在3000转（900 XG）离心1分钟洗两次的蠕虫。 </li><li>除去上清液，并添加M9与高达100-200微升。 </li><li>使用玻璃巴斯德吸管加一滴蠕虫/ M9混合以清除NGM板孵育它们至少24小时，在20℃，以允许胚胎孵化。 注：没有食物的幼虫的生长将在L1阶段停止。 </li></ol></li><li> 5日-建立同步L4幼虫人口 <ol><li>通过洗涤该板用1-2的M9毫升收集来自明确NGM板在L1幼虫，并将它们传送到一圆锥形15ml试管。 </li><li>让死成虫沉淀到15毫升锥形管的底部为约5分钟。 </li><li>收集上清液，其中所述L1蠕虫，在一个新的锥形15ml试管，并通过在2600转（1000 XG）离心2分钟沉淀出蠕虫。 </li><li>除去上清液乐AVING约500微升。 </li><li>通过滴用立体显微镜计数在10微升的蠕虫数目确定在M9溶液蠕虫的浓度。计数至少5滴。 </li><li>调整蠕虫的浓度为20-25蠕虫/微升，添加50微升的蠕虫/ M9混合物来接种OP50 NGM板。 </li><li>孵育65小时，在15℃（用于通过周末孵育），以允许在L1同步的人口增长，直到它们到达L4阶段。 </li></ol></li></ol> 4.暴露蠕虫在化学物质在96孔板注：本节介绍了使用Pxol1的含:: C.绿色荧光蛋白的转录记者线虫菌株筛选非整倍体的诱导。 <ol><li>通过洗涤该板用1-2的M9毫升收集来自明确NGM板的L4幼虫，并将它们传送到一圆锥形15ml试管。 </li><li>让L4蠕虫沉积物的底部的15毫​​升锥形管中大约5-10分钟。去除上清，而不会干扰病毒颗粒。 </li><li>加3-5的M9毫升，并确定由滴用立体显微镜计数在10微升的蠕虫数目，在M9溶液蠕虫的浓度。计数至少5滴每个样品。 </li><li>重悬在蠕虫中的M9 1000蠕虫/ ml的浓度。 </li><li>从稀释第1 OP50细菌10倍与M9。确保悬浮的细菌达到室温，因为较低的温度会影响蠕虫的发展。 </li><li>使用多道移液器，先添加100微升蜗杆/ M9混合物（来自步骤4.4）中，然后添加400微升的稀释OP50菌（来自步骤4.5），以对一个2毫升深圆底96孔板各孔中。在结束时，每个孔将具有在500μl100蠕虫。 </li><li>加入0.5微升的任一测试的化学品或适当的控制，以所期望的孔，以实现一个建议的最终精矿100μMATION，浓度一般在C.化学屏幕中使用线虫 12。暴露乙醇或DMSO溶剂对照为0.1％的最终浓度。 注意：我们建议使用诺考达唑（100μM）作为阳性对照。 </li><li>封用粘接膜的板。一定要密封板以及防止井之间的交叉污染。 </li><li>包装板用铝箔。 </li><li>板转移到适当的温度的摇床（170-180转）为适当长度的时间（24或65小时）。室内的温度影响的蠕虫的生长;维持20℃左右的温度下进行。 </li></ol>在384孔板5.图像采集妊娠蠕虫注意：这部分描述了使用具有高含量的显微镜图像的露出Pxol1 :: GFP蠕虫，可视化在成人雌雄同体的子宫内的胚胎的GFP的表达。为每384孔板进行筛选，用从4×96孔板蠕虫。 <ol><li>在摇床化学暴露后，让96孔板休息10-15分钟，以允许成虫沉降到板的底部。 </li><li>使用多道移液器，去除350微升的M9的，是非常小心，不要打扰蠕虫在板的底部。 </li><li> 1毫升M9和重复步骤5.1洗蠕虫。 </li><li>使用多道移液器，去除1毫升了M9的，是合租小心，不要打扰蠕虫在板的底部。 </li><li>使用多道移液器，悬浮蠕虫在剩余M9和收集100微升的蠕虫/ M9混合物从96孔板中，并将其加载到一个黑色壁/透明底384孔板的孔中。重复使用蠕虫从4×96孔板的过程中，直到已加载该384孔板的所有孔中。 注意：这是重要的，所有的孔中，以具有相同的体积，以便增加效率和自动聚焦的速度在图像采集过程中。每口井应该包含在80到100蠕虫。 </li><li>加入1μl左旋咪唑（100μM）的每个孔中，让蠕虫孵育大约30分钟。 注：左旋咪唑充当乙酰胆碱受体激动剂和固定不变的蠕虫。 </li><li>板块转移到能够提供自动成像的广角含量高的显微镜。 </li><li>进行图像采集，使用根据制造商的指导方针的高含量的图像采集和分析软件。选择4X目标收购每孔1的图像。 </li><li>启动图像采集前，调整GFP成像参数到45毫秒的曝光和图像分辨率为2,160 X 2,160的。 </li><li>收集数据作为GFP阳性蠕虫蠕虫的总数在井分割数，后者被测量井之间相对一致。 </li></ol>

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The authors declare that they have no competing financial interests.

此处所描述的方法构成第一大规模策略种系毒物的鉴定。它需要使用一个GFP转基因Pxol-1含有应变:: GFP的忠实报告非整倍体的诱导，其中用作种系功能障碍的代理早期胚胎。该方法涉及一个C的仔细同步线虫蠕虫人口和蠕虫暴露于化学品中的96孔格式接着GFP阳性蠕虫通过自动化高含量显微镜成像。 几个步骤这个协议是至关重要的结果的一致性。首先，?...

有在美国注册的商业约87000化学品，但只有一小部分，这些已经过测试，潜在的健康影响1。那些已被测试过的，仅一部分被评定为生殖健康的影响，部分原因是在确定改变哺乳动物早期生殖活动的困难，特别是在雌性生殖细胞发育和分化。事实上，第一减数分裂事件发生期间，在雌性哺乳动物胚胎发育的早期阶段，因此难以进入并收集在适于筛选目的号码。 生殖细胞提供了两代人之间的关键环节，其相应的功能是依赖于细胞和染色体分裂的复杂程序的精确执行称为减数分裂。减数分裂过程的失调可导致减少的生育力和生产配子和胚胎染色体数目异常，这种情况被称为非整倍体。减数分裂中染色体分离错误是高度相关的人体健康。染色体异常是常见的，具有一个频率的1在150个活产，21三体，18和13以及X和Y染色体的误差是最普遍的类型2,3。此外，先天性畸形，包括那些染色体来源的，是婴儿死亡的首要原因，在美国4中的环境影响可以影响染色体分离和行为的概念并不是新的5，但仍知之甚少。因此，关键是要考察其引入到我们的环境中的化学物质会干扰人的生育能力，早期的发展和整体生殖健康。 在光的哺乳动物模型的这些限制，我们开发了一种高通量筛选测定法中的蛔虫，以测试生殖毒性&lt;EM&gt;温度。线虫。我们已动员本常用的遗传模型系统提供了几个重要的功能，例如它的尺寸小，成本低，短的再现周期，生殖细胞的比例高，并易于操作6。蠕虫可以生长在96孔板或在高容量液体培养物并由于其透明性，可直接上成像板检测荧光报道的。下面所描述的实验利用了这些特点优势，以含荧光记者Pxol-1 :: GFP检测生殖干扰和诱导胚胎非整倍体的虫株的优势。 使用该报告菌株的是基于通常罕见的男性在一个主要雌雄同体蠕虫人口。这些男性（&lt;0.2％），自然地从错误中X染色体7的隔离起源。然而，由于种系破坏经常导致错误的常染色体的偏析heterochromosomes的和，它与雄性表型（Ⅹmissegregation）以及胚胎致死（常染色体missegregation）升高的发病率相关两者。容易地检测男性的感应而绕过胚胎致死的问题，一个雄性特异性启动子（XOL-1）用于驱动的GFP表达在早期胚胎仍含有蠕虫的子宫内。因此，表达GFP的胚胎的外观被用作用于非整倍体胚胎的存在的代理。此方法已被以前用于确定基因牵连种系维护和减数分裂8,9。适用于化学筛选，这株被采用在中高通量筛选。重要的是，应变忠实报告的化学品的aneugenicity，因此相关的哺乳动物的生殖端点10。这里描述的检测将是特别有用的毒理学家医药和化工等行业的设置看迅速评估朝生殖端点的化学品的毒性。此外，此法完全对准了毒性，在21 世纪11报告强调了政府的优先事项。

<table border="0" cellpadding="0" cellspacing="0" width="975">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Name of Reagent/ Equipment</td>
			<td style="width:129px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:255px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60mm vented, sharp edge Petri Dishes</td>
			<td style="width:129px;">Tritech</td>
			<td>T3315</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Agar</td>
			<td>Apex</td>
			<td>20-274</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:473px;">Axygen 96 Well Clear Round Bottom 2mL Polypropylene Deep Well Plate</td>
			<td style="width:129px;">Corning</td>
			<td>P-DW-20-C-S</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Bactopeptone</td>
			<td style="width:129px;">Apex</td>
			<td>20-261</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Bacto-tryptone</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP1421-2</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Bleach</td>
			<td style="width:129px;">Clorox</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride (CaCl2)</td>
			<td style="width:129px;">VWR</td>
			<td>AA12316-A1</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>ICN10138225</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">DMSO</td>
			<td style="width:129px;">VWR</td>
			<td>IC19018680</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">E. coli (OP50)</td>
			<td style="width:129px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Ethanol 200 proof</td>
			<td style="width:129px;">VWR</td>
			<td>EM-4455S</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Greiner CELLSTAR 384 well plates</td>
			<td>Sigma-Aldrich</td>
			<td>M1937-32EA</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageXpress Micro XLS System&#160;</td>
			<td style="width:129px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Levamisole hydrochloride&#160;</td>
			<td style="width:129px;">Fluka</td>
			<td style="width:119px;">31742</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate Anhydrous (MgSO4)</td>
			<td style="width:129px;">VWR</td>
			<td>97061-438</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MetaXpress High Content Image Acquisition &#38; Analysis Software&#160;</td>
			<td style="width:129px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Rayon Films for Biological Cultures</td>
			<td style="width:129px;">VWR</td>
			<td>60941-086</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:473px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S318</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:473px;">Sodium Phosphate Dibasic (Na2HPO4)</td>
			<td style="width:129px;">VWR</td>
			<td>BDH0316</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:473px;">Stereomicroscope&#160;</td>
			<td style="width:129px;">Nikon</td>
			<td style="width:119px;">SMZ 745</td>
			<td style="width:255px;">This microscope has a total magnification form 3.35x to 300x. Any microscope with similar characteristics will work.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:473px;">TY2441 C. elegans, Pxol-1::GFP reporter strain&#160;</td>
			<td style="width:129px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:473px;">Yeast extract</td>
			<td style="width:129px;">Becton Dickinson</td>
			<td style="width:119px;">212750</td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

comprehensive assessment of germline chemical toxicity using the nematode caenorhabditis elegans

识别数以千计的出现在我们的环境中的化学物质的生殖毒性已经在环境健康领域中最诱人的挑战之一。这部分对模型系统，可以（1）准确地概括生殖过程的键的功能和（2）这样做的一个中高通量方式的缺乏是由于，而不需要大量的脊椎动物的。 我们在这里描述的线虫C.化验线虫 ，它允许快速鉴定生殖系毒物通过监测非整倍体胚胎的诱导。通过利用GFP报告线，在染色体分离从种系的破坏而导致的错误很容易可视化并用自动荧光显微镜定量。由此一组特定于它的毒性化合物的筛选可以在96至384孔板格式在几天之内进行。积极H第二分析其可以被执行以确定是否染色体异常起源于减数分裂中断或从早期胚胎染色体分离错误。总之，此法代表了一个快速第一遍战略的生殖功能障碍的化学品接触的快速评估。

曝光Pxol-1 :: GFP报告菌株的化学试剂，如微管毒物诺考达唑（ ​​图1）导致了表达GFP的胚胎中相比DMSO对照露出成年雌雄同体的子宫高比例的诱导。 GFP阳性胚胎是比在其他胚胎以及自发荧光，在动物的肠道观察中观察到的弱的背景荧光显著亮。暴露蠕虫被直接成像在384孔板和蠕虫的含有至少一个GFP阳性胚胎的数量以及计算每个井和归由该蠕虫的总数。从化学屏幕正击中意味着诱...

Watch this Scientific Journal Video about Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans at JoVE.com

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

comprehensive-assessment-germline-chemical-toxicity-using-nematode

Research

JoVE Journal

Developmental Biology

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

8.0K 次观看.  University of California, Los Angeles. We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

视频: Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

基于发育毒性测定化学品安全筛选和系统生物学数据生成人类多能干细胞

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

科研

发育生物学

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

白银纳米胶体的盐度依赖性毒力测定使用青鳉鸡蛋

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein are molecular motors that regulate anterograde and retrograde transport, respectively. These motors use microtubule networks as rails. Caenorhabditis elegans (C. elegans) is a powerful model organism to study axonal transport and IFT in vivo. Here, I describe a protocol to observe axonal transport and IFT in living C. elegans. Transported cargo can be visualized by tagging cargo proteins using fluorescent proteins such as green fluorescent protein (GFP). C. elegans is transparent and GFP-tagged cargo proteins can be expressed in specific cells under cell-specific promoters. Living worms can be fixed by microbeads on 10% agarose gel without killing or anesthetizing the worms. Under these conditions, cargo movement can be directly observed in the axons and cilia of living C. elegans without dissection. This method can be applied to the observation of any cargo molecule in any cells by modifying the target proteins and/or the cells they are expressed in. Most basic proteins such as molecular motors and adaptor proteins that are involved in axonal transport and IFT are conserved in C. elegans. Compared to other model organisms, mutants can be obtained and maintained more easily in C. elegans. Combining this method with various C. elegans mutants can clarify the molecular mechanisms of axonal transport and IFT.

Immobilization of Caenorhabditis elegans to Analyze Intracellular Transport in Neurons

Caenorhabditis elegans (C. elegans) is a good model to study axonal and intracellular transport. Here, I describe a protocol for in vivo recording and analysis of axonal and intraflagellar transport in C. elegans.

固定化的线虫线虫分析神经元内细胞的转运

Axonal transport and intraflagellar transport (IFT) are essential for axon and cilia morphogenesis and function. Kinesin superfamily proteins and dynein ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are facilitated by the transparency of the animal, the ability to do forward and reverse genetic assays, the relative ease of generating fluorescently labeled proteins, and the use of fluorescent dyes that can either be microinjected into the early embryo or incorporated into its food (E. coli strain OP50) to label cellular organelles (e.g. 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1&#39;H-2,2&#39;-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron). Here, we present the use of a fluorescent pH-sensitive dye that stains intestinal lysosomes, providing a visual readout of dynamic, physiological changes in lysosomal acidity in live worms. This protocol does not measure lysosomal pH, but rather aims to establish a reliable method of assessing physiological relevant variations in lysosomal acidity. cDCFDA is a cell-permeant compound that is converted to the fluorescent fluorophore 5-(and-6)-carboxy-2&#39;,7&#39;-dichlorofluorescein (cDCF) upon hydrolysis by intracellular esterases. Protonation inside lysosomes traps cDCF in these organelles, where it accumulates. Due to its low pKa of 4.8, this dye has been used as a pH sensor in yeast. Here we describe the use of cDCFDA as a food supplement to assess the acidity of intestinal lysosomes in C. elegans. This technique allows for the detection of alkalinizing lysosomes in live animals, and has a broad range of experimental applications including studies on aging, autophagy, and lysosomal biogenesis.

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

A step-by-step guide to probe loss of lysosomal acidity in the intestine of C. elegans using the pH-sensitive vital dye 5(6)-carboxy-2&#39;,7&#39;-dichlorofluorescein diacetate (cDCFDA)

评估溶酶体碱化在活的秀丽线虫中的肠道

The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

种系线虫与胸苷模拟教育的细胞周期分析

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

线虫中耳炎群链球菌引起氧化应激的研究

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female reproductive tract. While in vitro studies suggest that sperm of internally fertilizing animals can respond to various cues from their surroundings, the inability to visualize their behavior inside the female reproductive tract creates a challenge for understanding sperm migration and mobility in its native environment. Here, we describe a method using C. elegans that overcomes this limitation and takes advantage of their transparent epidermis. C. elegans males stained with a mitochondrial&#160; dye are mated with adult hermaphrodites, which act as modified females, and deposit fluorescently labeled sperm into the hermaphrodite uterus. The migration and motility of the labeled sperm can then be directly tracked using an epi-fluorescence microscope in a live hermaphrodite. In wild-type animals, approximately 90% of the labeled sperm crawl through the uterus and reach the fertilization site, or spermatheca. Images of the uterus can be taken 1 h after mating to assess the distribution of the sperm within the uterus and the percentage of sperm that have reached the spermatheca. Alternatively, time-lapse images can be taken immediately after mating to assess sperm speed, directional velocity and reversal frequency. This method can be combined with other genetic and molecular tools available for the C.elegans to identify novel genetic and molecular mechanisms that are important in regulating sperm guidance and motility within the female reproductive tract.

Measuring Sperm Guidance and Motility within the Caenorhabditis elegans Hermaphrodite Reproductive Tract

Sperm must successfully navigate through the oviduct to fertilize an oocyte. Here, we describe an assay for measuring sperm migration within the C. elegans hermaphrodite uterus. This assay can provide quantitative data on sperm distribution within the uterus after mating, as well as on speed, directional velocity, and reversal frequency.

线虫生殖道内精子引导和运动的测定

Successful fertilization is fundamental to sexual reproduction, yet little is known about the mechanisms that guide sperm to oocytes within the female ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological conditions. Post-mitotic neurons represent a major cell type affected by aging. Although multiple mammalian models of neuronal aging exist, they are challenging and expensive to establish. The roundworm Caenorhabditis elegans is a powerful model to study neuronal aging, as these animals have short lifespan, an available robust genetic toolbox, and well-cataloged nervous system. The method presented herein allows for seamless isolation of specific cells based on the expression of a transgenic green fluorescent protein (GFP). Transgenic animal lines expressing GFP under distinct, cell type-specific promoters are digested to remove the outer cuticle and gently mechanically disrupted to produce slurry containing various cell types. The cells of interest are then separated from non-target cells through fluorescence-activated cell sorting, or by anti-GFP-coupled magnetic beads. The isolated cells can then be cultured for a limited time or immediately used for cell-specific ex vivo analyses such as transcriptional analysis by real time quantitative PCR. Thus, this protocol allows for rapid and robust analysis of cell-specific responses within different neuronal populations in C. elegans.

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Here, we present a protocol for simple isolation of specific groups of live neuronal cells expressing green fluorescent protein from transgenic Caenorhabditis elegans lines. This method enables a variety of ex vivo studies focused on specific neurons and has the capacity to isolate cells for further short-term culturing.

特定神经元种群与圆虫卡埃纳哈布迪炎的分离

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...