Name: using caenorhabditis elegans to screen for tissue-specific chaperone interactions
Uploaded: 2020-06-07T14:00:00.000+00:00
Duration: 6 min 55 s
Description: Watch this Scientific Journal Video about Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions at JoVE.com

我们感谢由NIH国家研究中心（NCRR）资助的卡诺哈布迪炎遗传学中心，感谢其一些线虫菌株。爱泼斯坦H.F.开发的单克隆抗体来自由NICHD主持、由爱荷华大学生物系维护的发展研究杂交瘤库。这项研究得到了以色列科学基金会的赠款（第278/18号赠款）和以色列科技部以及意大利共和国国家促进总局外交和国际合作部的赠款（第3-14337号赠款）的支持。我们感谢本-兹维实验室的成员帮助编写这份手稿。

1. 为 RNAi 准备线虫生长介质板
<ol><li>在 1 L 瓶中，加入 3 克 NaCl、2.5 克巴克托-佩普顿、17 克醋和蒸馏水至 1 L 和高压灭菌液。</li>
	<li>冷瓶至 55 °C。</li>
	<li>加入 25 mL 的 1 M KH2PO4、 pH 6.0、 1 mL 的 1 M CaCl2、 1 mL 的 1 M MgSO4和 1 mL 的胆固醇溶液 （表 1）来制作线虫生长介质 （NGM）。</li>
	<li>加入1 mL的安培素（100毫克/毫升）和0.5兆升的1M IPTG（表1），使NGM-RNAi解决方案。 注:常用的HT115（DE3） 大肠杆菌 含有一种IPTG诱导T7DNA聚合酶，用于表达dsRNA编码质粒。这些质粒还编码的安非他林抵抗。</li>
	<li>通过旋转瓶子混合温液。</li>
	<li>在发动机罩或使用无菌程序时，将 NGM 溶液倒入板中或使用渗透泵，将溶液分配到板中。使用 6 井、12 井、40 毫米或 60 mm 板来此屏幕。阿加应该填补约2/3的板深度。</li>
	<li>让盘子在室温下在长凳上干燥过夜，保持板材覆盖。RNAi 的 NGM 板可在 4 °C 下存储长达一个月。</li>
</ol>2. 种植RNAi细菌和播种板
<ol><li>在发动机罩或使用无菌程序时，将 1 mL 的氨基蛋白（100 毫克/毫升）和 2.5 mL 的四环素（5 毫克/毫升）（表 1）添加到自动封存前的 1 升 LB 溶液中并混合。 注:常用的HT115（DE3） 大肠杆菌 具有四环素耐药性。</li>
	<li>在发动机罩或使用无菌程序时，在 2 mL 深的 96 井无菌板中向每口油井添加 600 μL 的 LB 溶液。最好使用多通道管道来分配媒体。</li>
	<li>使用无菌程序，用HT115（DE3）大肠杆菌为油井接种疫苗，用dsRNA编码质粒转化，以感兴趣的基因或空质粒为控制。盖上盘子，在37°C的夜间孵育。由表达dsRNA的细菌克隆组成的库，相当于预测的C.elegans基因的94%，以前是22、23构建的，并且具有商业性。这里使用的陪护图书馆是由理查德·森本博士实验室26号建造的。</li>
	<li>使用无菌程序，种子75，150或250μL的细菌分别到12井，40毫米和6井或60毫米NGM RNAi板。清楚地标记盘子中目标基因的名称。细菌应覆盖30-50%的黄油表面，不应接触板的边缘。</li>
	<li>允许板在室温下在长凳上干燥至少 2 天，保持板材覆盖。 注:板块可在37°C过夜孵育。使用或存储板之前，请确保内井干燥。出于所有长期目的（即干燥或储存），将板保持在黑暗中。干燥的种子板可在 4 °C 下储存长达一个月。</li>
</ol>3. 胚胎无压力同步
<ol><li>使用蠕虫采摘将大约 100 个鸡蛋从未同步的蠕虫板移到新播种的 NGM 板。</li>
	<li>在 15 °C 下培养动物 5 天，在 20 °C 时培养动物 3.5 天，在 25 °C 下培养动物 2.5 天。 动物应该到产卵的第一天。 注:蠕虫通常在 20 °C 下培养。 然而，不同的伴郎突变菌株可能需要特定的栽培温度。例如，许多对温度敏感的菌株在 15 °C 下培养，但转移到 25 °C 以暴露其表型。</li>
	<li>缓慢地加入1兆L的M9缓冲器（表1），远离细菌草坪。旋转板，使缓冲器完全覆盖板。然后倾斜到一边，从盘子里取出液体，把动物从盘子里洗掉。 注:使用对温度敏感的动物或伴郎突变体时，最好在动物的培养温度下保持协议中使用的缓冲。</li>
	<li>重复步骤 3.3 三次或直到所有的动物被冲下盘子。</li>
	<li>使用标准塑料尖端，从鸡蛋集中的洗净板中切出一平方的醋，并将一块醋放入新播种的 NGM 板上。 注:需要200个鸡蛋才能生产出足够的产卵动物;太多的动物消耗细菌太快。低食物水平会影响蛋白石27。</li>
	<li>在 15 °C 下培养动物 5 天，在 20 °C 时培养 3.5 天，在 25 °C 下培养 2.5 天。 此时，盘子上应该覆盖着同步的鸡蛋。 注:动物可以转移到一个新的板短期更严格的同步。然而，重要的是，只有在产卵的早期阶段使用成人，因为动物可以保留卵子在他们的子宫影响同步。</li>
	<li>慢慢加入1mL的M9缓冲器，远离细菌草坪。</li>
	<li>旋转板，使缓冲器完全覆盖板。然后倾斜到一边，从盘子里取出液体，把动物从盘子里洗掉。</li>
	<li>重复步骤 3.7 三次或直到所有动物被冲出盘子。</li>
	<li>加入1兆L的M9缓冲器，并使用细胞刮刀从盘子中释放鸡蛋。</li>
	<li>从盘子中收集含有鸡蛋的 M9 缓冲器。</li>
	<li>将含有鸡蛋的 M9 缓冲器离心，在 3，000 x g 下 2 分钟。</li>
	<li>拆下超高音量并添加 M9 缓冲器，以达到 1 mL 的体积。</li>
	<li>重新悬念鸡蛋，以破坏任何块的鸡蛋和细菌。</li>
	<li>重复步骤 3.11-3.13 中描述的洗涤过程五次。蛋丸应该呈白色。如果它仍然是黄色/棕色，重复洗涤，直到达到白色颗粒。 注意:留在鸡蛋上的细菌会污染dsRNA表达细菌。</li>
	<li>去除大部分超高超， 留下大约 200 微升。同步鸡蛋可用于 Rnai 屏幕。</li>
</ol>4. 常见表型测定
<ol><li>实验期间动物的培养<ol>
<li>将一滴 +30 鸡蛋放在每个 RNAi 种子板的细菌草坪附近。供参考，也把+30鸡蛋放在盘子里，上面种着含有空载体（L4440）细菌的鸡蛋。</li>
		<li>在 NGM RNAi 种子板上培养年龄同步的动物。实验的持续时间将取决于监测动物的阶段和栽培温度。使用对温度敏感的突变动物时调整培养温度。使用发育迟缓的突变动物时调整培养时间。 注:虽然时间可能有所不同，但一旦动物成年，产卵（以及由此产生的快速食物消费），以及依赖年龄的蛋白体崩溃28，可能会影响结果。因此，建议在产卵开始前给动物打分（成年第1天）。野生动物在 15 °C 下 4.5 天后到达此阶段，在 20 °C 时达到 3 天，在 25 °C 下达到 2 天后达到此阶段。</li>
		<li>重复使用的数量将取决于所检查的基因集的大小。对于伴郎库（97个基因），重复实验至少四次。人口规模取决于所使用的检测。在此讨论的行为分析中，每次重复每次重复的实验情况得分>15。数据和统计分析也在很大程度上取决于使用的检测类型。此处讨论的测定中的数据可以作为 SEM ±表示。</li>
		<li>将RNAi和空载体控制处理动物作为独立种群进行比较。P 值可以使用单向或双向 ANOVA 计算，具体取决于所检查的变化，即单独或两者加重或减轻。在检查单个 RNAi 治疗与控制时，P 值可以使用单向或双向曼-惠特尼排名总和测试进行计算。除统计意义外，根据表型的影响程度考虑命中阈值。</li>
	</ol></li>
	<li>发展逮捕/拖延<ol>
<li>按照第4.1步培养年龄同步动物，直到在含有空载体的控制细菌上生长的动物成年，但在产卵开始前。</li>
		<li>使用立体显微镜监测动物，并计算幼虫和成人的数量，以得分发育迟缓的动物的百分比。供参考，与在空载体控制细菌上生长的突变动物相比。 hsp-1 或 hsp-90 RNAi 治疗导致野生动物发育性逮捕，可用作正控。</li>
		<li>要随着时间的推移，要对发育迟缓的动物进行评分，请重复步骤 4.2.2。 注:如果盘子里有产卵成人，将发育迟缓的动物转移到标有同一靶基因的新NGM-RNAi板上，以避免与后代混淆。</li>
	</ol></li>
	<li>不育或产卵缺陷<ol>
<li>培养年龄同步的动物，就像在第4.1步一样，直到在空病媒控制细菌上生长的动物开始产卵。</li>
		<li>使用立体显微镜监测动物，并给子宫内没有可见卵子的动物打分。供参考，比较在空病媒控制细菌上生长的突变动物。</li>
		<li>或者，使用立体显微镜监测动物，并给子宫内充满卵子的动物打分百分比，定义为EGG铺设缺陷（Egl-d）表型29。</li>
	</ol></li>
	<li>胚胎杀伤力<ol>
<li>培养年龄同步的动物，如第4.1步，直到动物开始产卵。</li>
		<li>将 +100 鸡蛋转移到空盘子中。将鸡蛋排成一排，以简化计数。</li>
		<li>24-48小时后，将盘子中未煮熟的鸡蛋的百分比评分。供参考，与用空病媒控制细菌治疗的动物的卵子进行比较。</li>
	</ol></li>
	<li>麻痹检测<ol>
<li>对于第 1 天成人，按照第 4.1 步培养年龄同步的动物，直到在空病媒控制细菌上生长的动物成年，但在产卵开始前。</li>
		<li>使用精细标记在普通 NGM 阿加板的背面画一条线。</li>
		<li>将 5-10 只动物放在标记线上。</li>
		<li>设置定时器并等待 10 分钟。</li>
		<li>将留在线上的动物的百分比评分为瘫痪蠕虫。供参考，比较在空病媒控制细菌上生长的突变动物。使用 unc-45 RNAi 治疗的野生动物表现出严重的瘫痪表型，可用作正控。 注:此检测突出显示中度至重度瘫痪的动物。这种动物通常直接躺在盘子上，而不是呈现常见的弯曲形状。此外，在瘫痪的蠕虫的头部周围可以看到清除细菌的斑块。</li>
	</ol></li>
	<li>鞭打分析<ol>
<li>按照第4.1步培养年龄同步动物，直到在空病媒控制细菌上生长的动物成年，但在产卵开始前。</li>
		<li>在动物的培养温度下将 100 μL 的 M9 缓冲液输送到 96 井板中。</li>
		<li>将 +15 蠕虫（每口井一只）放入含 M9 缓冲器的井中。</li>
		<li>让动物调整5分钟。</li>
		<li>在立体显微镜下检查每只动物，启动倒计时 15 s 的计时器，并计算每只动物在该时间段内进行的身体弯曲次数。计数的值可以正常化为每分钟身体弯曲。供参考，比较在空病媒控制细菌上生长的突变动物。 注:这种动感检测非常敏感，可以检测出治疗之间的非常轻微的差异。然而，液体中的动能和阿加的动能可能有所不同。</li>
	</ol></li>
</ol>5. 蛋白质击倒的验证
<ol><li>将 250-300 个同步鸡蛋放入 60 mm NGM-RNAi 板上，该板上播种了相关的 dsRNA 表达或空载体（L4440）细菌。 注:RNAi的击倒可能导致胚胎的异常积累，缺乏胚胎或发育性逮捕，这可能会影响基因表达。在确定要检查的动物的年龄时，应考虑这一点。</li>
	<li>对于第 1 天成人，在 15 °C 下培养动物 4.5 天，在 20 °C 时培养动物 3 天，在 25 °C 下养殖 2 天。</li>
	<li>挑选并转移总共 200 只年轻的成年动物到 1.5 mL 管的盖子中，填充 200 μL 的 PBS-T。 注:使用对温度敏感的动物或伴郎突变体时，最好在动物的培养温度下保持协议中使用的缓冲。</li>
	<li>小心地关闭盖子，离心机在 1，000 x g 下 1 分钟。</li>
	<li>加入 800 μL 的 PBS-T （表 1）和离心机在 1，000 x g 1 分钟。</li>
	<li>小心地删除顶部 900 μL。</li>
	<li>重复步骤 5.4-5.6 三次。</li>
	<li>取出 900 μL，留下 100 μL 的溶液，其中包含 200 蠕虫。</li>
	<li>添加 25 μL 的 5 倍样品缓冲器 （表 1）。</li>
	<li>在 92°C 下加热样品 10 分钟，同时在 1000 rpm 时摇晃。然后，样品可以冷冻并保存在 -20 °C 下。</li>
	<li>装载每个样品的 20 μL，并在 SDS-PAGE 凝胶上运行。</li>
	<li>使用适当的抗体进行西式斑点分析，以确定蛋白质的相对稳定性。</li>
	<li>使用密度测量软件（如免费提供的 ImageJ 凝胶模块）确定频段的强度。将所有值标准化到对照样本中测量值。 注:RNAi 在我们的屏幕上被击倒的目的是降低特定伴郎/伴郎的蛋白质水平。因此，评估 RNAi 击倒效率的最佳方法是通过西方污点分析。这需要特定的抗体。或者，qPCR 可用于量化 mRNA 水平。</li>
</ol>

作者没有什么可透露的。

一个反映它是如何组织起来的，并在不同的元子细胞和组织中发挥作用的蛋白石网络的综合图片仍然缺乏。为了解决这一缺陷，需要提供关于该网络各组成部分（如分子伴郎）在发育和老化过程中特定组织中相互作用的具体信息。在这里，我们展示了组织特异性扰动的使用如何使我们能够检查给定组织中的伴郎网络。为了探索组织特异性伴郎遗传相互作用，考虑了三种不同的方法。在第一种方法?...

细胞通过使用质量控制机器来修复、封存或去除任何受损的蛋白质1，2来应对蛋白质的损害。蛋白质复合物的折叠和组装由分子伴奏支持，这是一组高度保存的蛋白质，可以修复或封存受损的蛋白质3，4，5，6，7。去除受损蛋白质由无处不在的蛋白系统（UPS）8或自噬机械9与伴奏10，11，12合作进行调解。因此，蛋白质平衡（蛋白石）是由折叠和降解机3，13组成的质量控制网络维持的。然而，了解体内蛋白石网络各组成部分之间的相互作用是一项重大挑战。虽然蛋白质-蛋白质相互作用屏幕有助于提供关于物理相互作用和伴郎复合物的重要信息14，15，了解组织特定的伴郎网络在体内的组织和补偿机制是缺乏的。
基因相互作用经常被用作一种强大的工具，用来研究参与共同或补偿性生物通路16、17、18的基因对之间的关系。这种关系可以通过结合一对突变和量化一个基因突变对第二个基因16突变引起的表型严重性的影响来测量。虽然大多数此类组合在表型方面没有表现出任何效果，但某些遗传相互作用可能加剧或减轻所测量表型的严重程度。当双删除突变体的表型比组合单个删除突变体时看到的预期表型更严重时，观察到加重突变，这意味着两个基因在平行通路中工作，共同影响给定函数。相比之下，当双删除突变体的表型不如单个删除突变体中出现的表型严重时，则观察到缓解突变，这意味着两个基因作为一个复合体协同作用或参与同一通路16、18。因此，可以量化的多种表型，包括广泛的表型，如致命性、生长率和育雏大小，以及特定表型（如转录记者）已用于识别遗传相互作用。例如，Jonikas等人依靠ER应激记者使用配对基因删除分析19来检查糖核酸细胞ER展开蛋白反应蛋白体网络的相互作用。
基因相互作用屏幕涉及系统地交叉配对删除突变，以产生一套全面的双突变体20。然而，在动物模型中，特别是在C.elegans中，这种大规模的方法是不可行的。相反，突变菌株可以通过使用RNA干扰（RNAi）21对基因表达进行向下调节来测试其遗传相互作用模式。C. elegans是一个强大的系统，基于 RNAi22，23屏幕。在C.elegans中，双链RNA（dsRNA）的传递是通过细菌喂养实现的，导致dsRNA分子扩散到许多组织。通过这种方式，引入的dsRNA分子通过快速和简单的程序21影响动物。因此，使用RNAi的基因相互作用屏幕可以揭示使用RNAi库24对一组基因或大多数C.elegans编码基因进行下调节的影响。在这样的屏幕上，影响感兴趣的突变体的行为，但不是野生类型菌株的命中是被监测的表型的潜在修饰剂25。在这里，我们应用突变和RNAi筛选的组合，系统地绘制C.elegans组织特异性伴郎相互作用的地图。

<table><tbody><tr><td>12-well-plates</td><td>SPL</td><td>BA3D16B</td><td></td></tr><tr><td>40 mm plates</td><td>Greiner Bio-one</td><td>627160</td><td></td></tr><tr><td>60 mm plates</td><td>Greiner Bio-one</td><td>628102</td><td></td></tr><tr><td>6-well plates</td><td>Thermo Scientific</td><td>140675</td><td></td></tr><tr><td>96 well 2 mL 128.0/85mm</td><td>Greiner Bio-one</td><td>780278</td><td></td></tr><tr><td>Agar</td><td>Formedium</td><td>AGA03</td><td></td></tr><tr><td>Ampicillin</td><td>Formedium</td><td>69-52-3</td><td></td></tr><tr><td>bromophenol blue</td><td>Sigma</td><td>BO126-25G</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>1.02382.0500</td><td></td></tr><tr><td>Camera</td><td>Qimaging</td><td>q30548</td><td></td></tr><tr><td>Cholesterol</td><td>Amresco</td><td>0433-250G</td><td></td></tr><tr><td>Confocal</td><td>Leica</td><td>DM5500</td><td></td></tr><tr><td>Filter (0.22 &amp;micro;m)</td><td>Sigma</td><td>SCGPUO2RE</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>Leica</td><td>MZ165FC</td><td></td></tr><tr><td>Glycerol</td><td>Frutarom</td><td>2355519000024</td><td></td></tr><tr><td>IPTG</td><td>Formedium</td><td>367-93-1</td><td></td></tr><tr><td>KCl</td><td>Merck</td><td>104936</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>1.04873.1000</td><td></td></tr><tr><td>KOH</td><td>Bio-Lab</td><td>001649029100</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher</td><td>22189-08-8</td><td>Gift from the Morimoto laboratory</td></tr><tr><td>Myosin MHC A (MYO-3) antibody</td><td>Hybridoma Bank</td><td>5-6</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>s-0751</td><td></td></tr><tr><td>NaCl</td><td>Bio-Lab</td><td>001903029100</td><td></td></tr><tr><td>Peptone</td><td>Merck</td><td>61930705001730</td><td></td></tr><tr><td>Plate pouring pump</td><td>Integra</td><td>does it p920</td><td></td></tr><tr><td>RNAi Chaperone library</td><td>NA</td><td>NA</td><td></td></tr><tr><td>SDS</td><td>VWR Life Science</td><td>0837-500</td><td></td></tr><tr><td>&amp;szlig;-mercaptoethanol</td><td>Bio world</td><td>41300000-1</td><td></td></tr><tr><td>stereomicroscope</td><td>Leica</td><td>MZ6</td><td></td></tr><tr><td>Tetracycline</td><td>Duchefa Biochemie</td><td>64-75-5</td><td></td></tr><tr><td>Tris</td><td>Bio-Lab</td><td>002009239100</td><td></td></tr><tr><td>Tween-20</td><td>Fisher</td><td>BP337-500</td><td></td></tr></tbody></table>

using caenorhabditis elegans to screen for tissue-specific chaperone interactions

蛋白质和蛋白质复合物的正确折叠和组装对细胞功能至关重要。细胞采用质量控制途径，纠正、封存或消除受损蛋白质，以维持健康的蛋白质组，从而确保细胞蛋白石和防止进一步的蛋白质损伤。由于蛋白酶网络中的多余功能，使用多细胞生物 体Caenorhabdd炎的 伴随编码基因的敲击或突变来筛选可检测表型，在大多数情况下，可检测到小或无表型。我们制定了有针对性的筛选策略，以确定特定功能所需的陪护人员，从而弥合表型和功能之间的差距。具体来说，我们使用RNAi合成交互屏幕监测新的伴郎互动，敲击伴郎表情，一次一个伴郎，在携带伴郎编码基因突变或过度表达兴趣伴郎的动物中。通过破坏两个单独呈现无严重表型的陪护，我们可以识别在两者都扰动时加重或暴露特定表型的陪护。我们证明，这种方法可以识别出特定的伴郎组合，这些伴郎可以协同工作，调节与给定表型相关的蛋白质或蛋白质复合物的折叠。

分别使用 UNC-45 中的温度敏感突变来筛选在允许或限制条件下加重或减轻相互作用的情况 肌肉组装和维护提供了一个有效的系统来研究组织特定的伴郎相互作用。收缩肌肉的功能单元，肉瘤，呈现一个水晶般的结构和调节蛋白排列。运动蛋白肌素的稳定性及其融入收缩肌瘤的厚丝取决于伴郎和UPS组件30的合作。一个例子，这样的陪护是保存和专门肌素伴奏UNC-45，主...

Watch this Scientific Journal Video about Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions at JoVE.com

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

为了研究伴奏和伴奏基底相互作用，我们使用RNA干扰与轻度突变或伴郎过度表达相结合，在有机体水平上监测组织特异性蛋白质功能障碍，在 卡诺哈布德炎中 执行合成相互作用屏幕。

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

using-caenorhabditis-elegans-to-screen-for-tissue-specific-chaperone

Research

JoVE Journal

Biology

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

2.8K 次观看.  Ben-Gurion University of the Negev. 为了研究伴奏和伴奏基底相互作用，我们使用RNA干扰与轻度突变或伴郎过度表达相结合，在有机体水平上监测组织特异性蛋白质功能障碍，在 卡诺哈布德炎中 执行合成相互作用屏幕。

视频: Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

科研

生物化学

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

癌症研究

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

发育生物学

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

高通量筛选和生物传感与荧光 C。线虫菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

生物学

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

神经科学

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物<em&gt;温度。线虫</em

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

可筛选的<em&gt;在体内</em&gt;测定线粒体调节剂使用转基因生物发光<em&gt;秀丽隐杆线虫</em

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

使用卡诺哈布迪炎埃莱甘斯筛选组织特异性查佩龙相互作用

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一种简单的高通量化学筛选方法在秀丽线虫中的应用

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

高通量筛选和生物传感与荧光 C。线虫菌株

基本的

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物

可筛选的

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

使用 卡诺哈布迪炎埃莱甘斯 筛选组织特异性查佩龙相互作用

摘要

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此视频中的章节

一种简单的高通量化学筛选方法在秀丽线虫中的应用

使用卡诺哈布迪炎埃莱甘斯识别 EGFR 和 RAS 抑制剂

来自卡诺哈布迪炎精英的蛋白质提取制剂制备和共免疫沉淀

高通量筛选和生物传感与荧光 C。线虫菌株

基本的

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

调查的传播和毒性朊病毒样蛋白使用后生动物模式生物

可筛选的

量化秀丽隐杆线虫中组织特异性蛋白静剂下降

使用卡诺哈布迪炎埃莱甘斯筛选组织特异性查佩龙相互作用

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统