我们要感谢 秀丽隐杆线 虫社区的实验室，他们开发和分享了这些工具，他们的工作在本手稿中被引用。一些菌株由CGC提供，CGC由NIH研究基础设施计划办公室（P40 OD010440）资助。这项工作得到了加拿大卫生研究院（CIHR）对ALS项目资助（PJT-175245）的支持。CL 由加拿大自然科学与工程研究委员会 （NSERC） 研究生奖学金 （PGS-D） 支持。

注：检测时间线如图 1所示。
1. RNAi线虫生长培养基（RNAi-NGM）平板的制备
<ol><li>将含有GFP或对照RNAi载体的大 肠杆菌 HT115（DE3）从甘油储备液中划出到补充有100μg/ mL氨苄青霉素的Luria-Bertani（LB）琼脂平板上。将细菌在37°C孵育过夜。</li>
	<li>第二天，根据标准方案18制备RNAi-NGM板（1.7%[w/v]琼脂，0.3%[w/v]NaCl，0.25%[w/v]蛋白胨，1mM CaCl 2,5μg/ mL胆固醇，25mM磷酸钾缓冲液[pH 6.0]，1mM MgSO4,25μg/ mL羧苄青霉素和5mM异丙基β-d-1-硫代吡喃半乳糖苷[IPTG]）。<ol><li>对于测定中的每种菌株，准备至少六个接种有GFP RNAi的RNAi-NGM板（三个生物学重复中的每一个两个板）和两个接种对照RNAi的RNAi-NGM板（一个生物学重复）。</li>
		<li>用箔纸松散地将盘子包裹起来以避光，并在室温下放置过夜晾干。</li>
	</ol></li>
	<li>在 RNAi-NGM 平板倾倒的同一天，制备 4 mL RNAi 细菌的液体培养物。<ol><li>在无菌条件下，将补充有 10 μg/mL 四环素和 100 μg/mL 氨苄青霉素的 4 mL LB 肉汤分装到培养管中。将LB琼脂平板中的单个菌落选入每个管中，并在37°C下振荡约16小时孵育过夜。</li>
	</ol></li>
	<li>将RNAi细菌接种到RNAi-NGM板上。<ol><li>过夜生长后，使用LB肉汤以1：5稀释的细菌测量培养物的光密度（OD600）。</li>
		<li>使用补充有 10 μg/mL 四环素和 100 μg/mL 氨苄青霉素的 LB 肉汤将 RNAi 细菌稀释至相对 OD600 为 2。</li>
		<li>在无菌条件下，将 150 μL 对照或 GFP RNAi 细菌加入每个 RNAi-NGM 板上。</li>
		<li>使用前用箔纸覆盖板，让细菌在室温下生长至少24小时。 注意：未使用的RNAi-NGM板可以在4°C下在黑暗的密封容器中倒置储存长达1周。</li>
	</ol></li>
</ol>2. 开始RNAi遗传测定：漂白和接种P0代胚胎
注意：在开始RNAi遗传测定之前，含有GFP报告基因mjIs134 [mex-5p：：gfp-h2b：：tbb-2 3'UTR]7 的蠕虫应在21°C下保持至少两代。
<ol><li>在RNAi遗传测定开始前4天（96小时）（图1），将三个或四个妊娠成虫选入每个接种有OP50-1细菌的35mm标准NGM板上。 注意：每个菌株三个NGM板就足够了。对于生育能力受损的菌株，每块板可能需要四只以上的动物。</li>
	<li>4 天后，确保成年人群已开始在培养皿上产下胚胎。使用补充有Triton X-100（TX-100）（22mM KH 2 PO 4,42mM Na2HPO 4,86mM NaCl，1mMMgSO 4,0.01%（v / v）TX-100）的800μLM9缓冲液将蠕虫从板上洗入1.5mL管中。重复洗涤并池入同一管中。</li>
	<li>通过漂白将胚胎从群体中分离出来，如下所示。<ol><li>用漂白剂（6%次氯酸钠）和10N NaOH以1.5：1的体积比制备漂白溶液。准备足够的量，以便每个样品可以使用 250 μL。</li>
		<li>将 1.5 mL 管与蠕虫以 1,000 x g 离心 1.5-2 分钟。</li>
		<li>吸出上清液，留下 100 μL 而不干扰蠕虫“沉淀”。或者，可以将管子放置约2分钟，以使蠕虫在吸气前沉淀。</li>
		<li>向每个试管中加入 650 μL ddH2O，以达到 750 μL 的最终体积。 注意：以下步骤具有时效性。</li>
		<li>向每个试管中加入 250 μL 漂白溶液并启动计时器。</li>
		<li>每1-2分钟，彻底涡旋试管。5分钟后，检查立体镜下的蠕虫，以监测成虫的降解。</li>
		<li>继续涡旋，直到蠕虫完全溶解，只剩下胚胎。立即将试管以1,000× g 离心1.5分钟。 注意：漂白通常在6-7分钟内完成，但应在立体镜下以足以观察释放的胚胎（40x）的放大倍率进行监测。不要将蠕虫长时间放在漂白剂中，因为这会损害胚胎。</li>
		<li>离心后，吸出上清液并留下约 50-100 μL。 加入 1 mL 含 TX-100 的 M9 缓冲液洗涤并通过涡旋混合。再次以1,000× g 离心1.5-2分钟，再洗涤至少两次。</li>
		<li>从最终洗涤中吸出上清液，并在试管中留下约 100 μL。涡旋混合。将 2 μL 从每个试管移液两次到标记的载玻片上。使用计数器对胚胎进行计数，以估计每微升胚胎的浓度。 注意：此处可以使用具有多个磨砂孔的载玻片来计数多个菌株的重复。计数载玻片可以清洗和重复使用。</li>
	</ol></li>
	<li>为了开始半同步的种群增长，将含有250个胚胎的体积混合并移液到每个RNAi-NGM板上。每次重复使用两个板。让液体吸收。</li>
	<li>对于用RNAi处理的P0世代，在21°C孵育4天（96小时）（从胚胎到成年）。时间可能因基因型而异。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65285/65285fig01.jpg"> 图1：RNAi遗传检测示意图。 RNAi-NGM 板制备和 RNAi 遗传测定设置的建议时间表。在第 -4 天将妊娠成虫采摘到接种有 OP50-1 的 NGM 平板上。4天后，将成年后代漂白，并将胚胎接种到RNAi-NGM平板上。将P0世代在21°C下暴露于RNAi中4天。 一旦蠕虫达到成年期，就会通过漂白对重复进行传代，并在每一代中对种系GFP表达进行评分。 <a href="https://www.jove.com/files/ftp_upload/65285/65285fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
3. 种系GFP表达每一代的传代和评分
注意：为了便于评分，请使用黑胶唱片创建具有线性脊的琼脂糖垫以排列蠕虫。这种方法改编自 Rivera Gomez 和 Schvarzstein19。
<ol><li>通过在微波炉中加热将琼脂糖溶解在小烧瓶中的ddH2O中，制备1%（w / v）琼脂糖。加入搅拌棒并用箔纸盖住。如果重熔，在搅拌的同时在200°C的加热板上加热琼脂糖。熔化后，将热量降低至80°C。</li>
	<li>使用 800 μL 含 TX-100 的 M9 缓冲液将蠕虫从 NGM 板上洗净到 1.5 mL 管中。清洗板两次以去除所有妊娠成虫。检查立体镜下的板，以确认动物已被有效收集。</li>
	<li>将试管以1,000× g 离心1.5-2分钟或让蠕虫沉降2分钟。吸出上清液并留下 100 μL，而不会干扰蠕虫“沉淀”。</li>
	<li>准备琼脂糖垫以安装蠕虫，如下所示。<ol><li>使用玻璃巴斯德移液管（窄端折断）将一滴熔化的1%（w / v）琼脂糖滴到黑胶唱片（前面描述19）上。</li>
		<li>定向载玻片，使记录上的线条水平或垂直，然后将载玻片快速放在琼脂糖液滴上。等待大约 30 秒，然后从记录中取出载玻片。 注意：如果对多种基因型进行评分，则在一张载玻片上制作更大的琼脂糖垫并使用刀片将其切成两半可能很有用。</li>
	</ol></li>
	<li>将蠕虫安装到琼脂糖垫上。<ol><li>在立体镜下，向琼脂糖垫中加入约5μL的5mM左旋咪唑。左旋咪唑稀释液应每周新鲜。</li>
		<li>轻轻轻弹蠕虫管，将 5-10 μL 蠕虫混合并转移到琼脂糖垫上。在添加蠕虫时通过肉眼估计蠕虫的数量，以便每次重复大约有 40 个蠕虫。</li>
		<li>使用睫毛镐将蠕虫排成一排，以便于评分。添加玻璃盖玻片。 注意：以这种方式安装动物的载玻片可以持续几个小时，然后才会变干。</li>
	</ol></li>
	<li>在对载玻片进行评分之前，使用漂白方案（参见步骤2.3）将胚胎与剩余的动物分离。将 250 个胚胎接种到 35 mm NGM 平板上，接种有 OP50-120。一旦液体被吸收，倒置板并将其放回21°C培养箱中。</li>
	<li>使用带有 GFP 滤光片组的荧光立体镜。使用计数计数器计数并记录每次重复的载玻片上GFP阳性和GFP阴性蠕虫的数量。 注意：GFP阳性蠕虫在其生殖系和 子宫内胚胎中具有核GFP表达。GFP阴性蠕虫的种系不会发出荧光，但是，随着GFP在后代中开始重新打开，荧光可能会变暗。安装额外的非转基因蠕虫菌株以解释观察到的任何自发荧光可能会有所帮助。</li>
	<li>如上所述，在21°C下大约每4天（96小时）漂白一次。 或者，为方便起见，可以按 3 天/4 天交替的时间表进行传代。如果交替进行，则为较短的传代接种额外的~50个胚胎，因为72小时后胚胎的产量可能会降低。 注意：在胚胎铺板后 4 天保持 P0 代传代时间一致。</li>
</ol>4. ChIP 动物采集
注：动物数量和时间取决于菌株、发育阶段、表位和免疫沉淀 （IP） 靶标的数量。在下面的示例中，收集三个 IP 的动物：H3K9me3、组蛋白 H3 和 IgG 对照。ChIP 方案改编自 Askjaer 等人 21。
<ol><li>在 ChIP 样本采集之前，将上一代 RNAi 遗传检测再增加 3 个 NGM 板（每次重复至少增加 4 个板）。在21°C下生长4天。</li>
	<li>漂白妊娠成虫以分离胚胎（参见步骤2.3）。</li>
	<li>对于每个菌株，将大约3,500个胚胎铺在14个平板上（每块平板250个），并在21°C下生长3天至年轻成人阶段。</li>
	<li>用含有0.01%（v / v）TX-100（PBS / TX）的磷酸盐缓冲盐水（PBS）将动物洗涤到1.5mL管中。以1,000× g 离心2分钟。将动物从一个菌株中吸出并汇集到一个管中，并用至少 1 mL PBS/TX 再洗涤三次。</li>
	<li>吸出至1,000μL，倒置混合，并计数3μL中的动物数量3次（参见步骤2.3.9）。</li>
	<li>计算动物的浓度，并将相当于3,000只动物的体积转移到新管中进行甲醛交联。</li>
	<li>确保总体积至少是蠕虫颗粒体积的 10 倍。</li>
</ol>5.甲醛交联
注意： 在通风橱中使用甲醛以防止接触蒸汽。
<ol><li>将甲醛（1.8%终浓度）添加到装有蠕虫的试管中。在室温下旋转 6 分钟。</li>
	<li>立即在液氮中冷冻。实验可以在此步骤暂停，样品可以储存在-80°C。</li>
	<li>将交联样品在室温水浴中解冻3分钟，并在室温下旋转16分钟。</li>
	<li>加入1.25M甘氨酸至终浓度为125mM，旋转5分钟。 注意：在磁珠洗脱之前，将样品保持在冰上或4°C，并使用冰冷的缓冲液。</li>
	<li>将样品以1,000× g 离心3分钟。每次用 1 mL PBS/TX 洗涤 3 次。用 1 mL 重悬缓冲液（150 mM NaCl、50 mM HEPES-KOH [pH 7.5]、1 mM 乙二胺四乙酸 [EDTA]、0.01% TX-100、蛋白酶抑制剂 [每 5 mL 一片]）洗涤两次。</li>
	<li>最后一次洗涤后，留出足够的缓冲液，以确保总体积至少是沉淀体积的三倍，并且至少为 ~100 μL。</li>
</ol>6. 超声处理
注意：超声处理参数取决于超声仪的类型和型号以及动物阶段。样品体积和浓度、开/关间隔、循环次数和功率设置等参数需要根据经验进行优化。例如，在超声处理的时间过程中，使用蛋白质测定法监测蠕虫裂解，并确定浓度何时达到平台期。此外，通过在1.5%琼脂糖/三乙酸酯-EDTA（TAE）凝胶上电泳DNA进行电泳，监测基因组DNA的平均剪切大小何时约为200-1,000 bp。
<ol><li>测量上一步的样品体积。将 90-120 μL 混合并分装到聚苯乙烯超声处理管中。</li>
	<li>加入等体积的含有 2x 去污剂（150 mM NaCl、50 mM HEPES-KOH [pH 7.5]、1 mM EDTA、0.2% 脱氧胆酸钠、0.7% sarkosyl）的重悬缓冲液。</li>
	<li>在4°C下以50%功率在水浴超声仪中超声处理7分钟（20秒开/40秒关）。 通过移液轻轻混合。重复超声处理7分钟。</li>
	<li>将超声处理的裂解物转移到 1.5 mL 试管中。加入 0.5 体积不含去污剂的重悬缓冲液（150 mM NaCl、50 mM HEPES-KOH [pH 7.5]、1 mM EDTA）（例如，向 200 μL 样品中加入 100 μL 缓冲液）。</li>
	<li>在4°C下以13,000× g 离心15分钟。 保留裂解物上清液并转移到新管中。</li>
	<li>或者，进行蛋白质测定以确定每种裂解物的浓度。此步骤可用于大样本量或批间比较。然而，如上所述标准化动物数量适用于此处描述的样品量，因为与qPCR测定的染色质/ DNA产量具有更好的相关性。</li>
</ol>7. 免疫沉淀
注：将抗体和磁珠的量与裂解物的体积和浓度成比例。
<ol><li>将裂解物上清液分成四份：每个 IP 三个相等体积，一个 IP 体积的 10% 作为起始量（例如，100 μL IP #1、100 μL IP #2、100 μL IP #3、10 μL 起始量）。将IP裂解物转移到200μL PCR管中，并将输入裂解物在-20°C下储存在1.5mL管中。</li>
	<li>向适当的 IP 样品中加入 0.5 μg 抗 H3K9me3 抗体、抗组蛋白 H3 抗体或 IgG。在4°C下旋转孵育过夜。</li>
	<li>第二天，将 9 μL 蛋白 G 包被的磁珠（每个 IP 分装到单个 1.5 mL 试管中）中。用 1 mL FA-150（150 mM NaCl、50 mM HEPES-KOH [pH 7.5]、1 mM EDTA、1% TX-100、0.1% 脱氧胆酸钠）洗涤珠子两次。</li>
	<li>将FA-150缓冲液中的磁珠重悬至上述步骤7.3中从原液中取出的原始体积。向每个 IP 中加入 7.5 μL。在4°C下旋转孵育2小时。</li>
</ol>8. 洗涤和洗脱
注意：为确保磁珠不会变干，请在吸出前一次洗涤后快速添加每次洗涤或洗脱缓冲液。
<ol><li>每次使用 0.2-1 mL 以下缓冲溶液洗涤珠子。将珠子收集在磁性支架上以吸出洗涤液。旋转将每次洗涤在4°C孵育5分钟。请按照以下顺序操作。<ol><li>用 FA-150 洗涤两次。</li>
		<li>用FA-1M洗涤一次（FA-150用1M NaCl洗涤一次）。</li>
		<li>用FA-0.5M洗涤一次（FA-150用0.5M NaCl洗涤一次）。转移到新的PCR管中。</li>
		<li>用TE-LiCl（250mM LiCl，10mM Tris-Cl [pH 8.0]，1mM EDTA，1%IGEPAL CA-630,1%脱氧胆酸钠）洗涤一次。</li>
		<li>用TE+（50mM NaCl，10mM Tris-Cl [pH 8.0]，1mM EDTA，0.005%IGEPAL CA-630）洗涤两次。 注意：除非另有说明，否则在室温下继续样品处理。</li>
	</ol></li>
	<li>吸出最后的洗涤缓冲液。用 50 μL ChIP 洗脱缓冲液（200 mM NaCl、10 mM Tris-Cl [pH 8.0]、1 mM EDTA、1% 十二烷基硫酸钠 [SDS]）重悬磁珠，并转移到 1.5 mL 试管中。</li>
	<li>在热混合器中在65°C洗脱15分钟，每分钟以1,000rpm混合5秒。</li>
	<li>将珠子收集在磁性支架上，并将上清液转移到新管中。</li>
	<li>用另外 50 μL ChIP 洗脱缓冲液重复洗脱。将上清液合并至总计 100 μL。 继续进行反向交联和 DNA 洗脱，如下所述。</li>
</ol>9. 反向交联和DNA洗脱
<ol><li>解冻输入裂解物样品。用 ChIP 洗脱缓冲液加满至 100 μL。</li>
	<li>向每个 IP 和输入样品中加入 16.5 μg RNase A。在37°C孵育1小时。</li>
	<li>加入40μg蛋白酶K，在55°C孵育2小时。然后，在65°C孵育过夜。</li>
	<li>将样品冷却至室温，并用离心柱试剂盒纯化DNA。</li>
</ol>10. qPCR反应的建立和运行
注：应修改引物、反应设置和热循环仪参数，以符合制造商对所用qPCR反应混合物的建议。
<ol><li>制备靶向 GFP RNAi 报告基因和 H3K9me3 阳性和阴性富集对照区域的引物组。底漆熔融温度为60°C。 引物序列见 材料表 。</li>
	<li>对于起始 DNA，进行四次四倍连续稀释（例如，1：5、1：20、1：80、1：320）。 注：IP DNA 可直接使用或稀释（例如，1：2 或 1：3）以允许更多反应。稀释液的适用性必须根据经验确定，因为qPCR性能可能会受到影响。</li>
	<li>在一个PCR板上组织与一组引物相对应的所有反应。为每个起始 DNA 稀释液和每个 IP DNA 样品设置技术重复反应。每个 10 μL 反应包含 1.5 μL 输入或 IP DNA（或洗脱缓冲液作为对照）、qPCR 预混液（1x 终浓度）、正向引物和反向引物（每个引物终浓度为 400 nM）。</li>
	<li>在实时热循环仪上，运行以下程序：初始变性：95°C4分钟;扩增和荧光检测：95°C10秒和60°C30秒的40个循环，板读数;最终延伸：60°C5分钟;熔体曲线：从 60 °C 到 90 °C，增量为 0.5 °C，每步 5 秒。</li>
</ol>11. 确定扩增效率并验证产品特异性
<ol><li>对于每组输入 DNA 稀释度，在 x 轴上用 [log10（1/dilution）] 绘制四个数据点，在 y 轴上用 [input Cq] 绘制。确定最佳拟合线的斜率。</li>
	<li>计算扩增效率。理想的引物组应始终显示 95%-100% 的效率。 <img alt="Equation 1" src="/files/ftp_upload/65285/65285eq01.jpg" style="margin: auto;"></li>
	<li>检查所有反应是否都有一个尖锐的熔融曲线峰，并且具有相同引物组的反应是否具有相同的熔融温度。多个峰或不同的熔解温度可能表明非特异性扩增。</li>
	<li>或者，在室温下在标准 2% 琼脂糖/TAE 凝胶上进行反应，以验证产物条带大小。</li>
</ol>12. 计算输入百分比
<ol><li>计算输入稀释因子。由于 IP 裂解物体积的 10% 保存为输入，因此 1：5 稀释输入 DNA 的稀释因子为 50。</li>
	<li>确定IP稀释因子。如果在qPCR之前未稀释IP DNA，则稀释因子为1。</li>
	<li>计算 IP 和输入之间的 Cq 差值，根据稀释因子进行调整。 <img alt="Equation 2" src="/files/ftp_upload/65285/65285eq02.jpg" style="margin: auto;"></li>
	<li>计算输入的百分比。 <img alt="Equation 3" src="/files/ftp_upload/65285/65285eq03.jpg" style="margin: auto;"></li>
</ol>

秀丽隐杆线虫的表观遗传遗传和 ChIP

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所有作者都确认他们没有任何冲突要披露。

在该方案中，通过喂养引入dsRNA，这已成为秀丽隐杆线虫18中的标准方法。对于RNAi遗传测定，补料方法提供了一种获得大P0群体2,11,12,22,23,24,25的简单方法。然而，RNAi暴露的时间和持续时间会影响转基因沉默的功效26，RNAi细菌的浓度会影响可遗传RNAi沉默的持久性1。因此，标准化的RNAi细菌和蠕虫生长对于获得一致的GFP沉默水平和遗传持续时间非常重要。在这里，胚胎被接种在RNAi平板上，以便P0动物在孵化时暴露于RNAi细菌。替代方法是将同步的 L1 24、27 或L4 25 期动物接种到 RNAi-NGM 平板上。此外，由于饥饿和其他应激会影响RNAi遗传13的维持，因此必须监测平板以防止过度拥挤和食物供应枯竭。作为通过喂养启动RNAi的替代方法，一些开创性的秀丽隐杆线虫RNAi遗传研究通过性腺注射诱导RNAi，从而更好地控制dsRNA浓度1,28。
在该方案中，每一代都作为碱性次氯酸盐处理的群体传代，如前所述16,22,23,24。次氯酸盐处理可确保 F1 生成不会被来自亲本环境的 RNAi 细菌污染22，并防止潜在的不良种群瓶颈。然而，批量传代也可能是一个限制，因为个体动物可能具有不同的遗传模式1,29。建立每一代的另一种方法是选择个体动物1、2、9、11、25。这种方法允许跟踪谱系内的表型和遗传杂交的结合。谱系分析也可能对低外显率表型有利12.
荧光蛋白表达是 RNAi 诱导的沉默2、3、4、12、14、15、16、25、30 的强大而方便的读数。如该协议所述，GFP表达可以手动评分为ON或OFF 11,12,15,16,25。手动评分可以通过分配定性强度级别3、4、14 来进一步完善。或者，显微镜图像11、12、14、25、27的自动强度评分或活体动物2的流式细胞术荧光测量可以提供定量和高通量读数。然而，由于报告基因表达仅限于种系，因此自动方法的一个警告是，它们还必须区分GFP表达沉默的动物和没有种系的动物，特别是如果研究包含具有异常种系发育的突变体。作为 GFP 荧光的替代方法，逆转录 （RT）-qPCR 可用于定量 RNAi 靶标前 mRNA 和 mRNA 水平 2,16,23,24。这种方法提供了更直接的沉默读数，其靶向RNA，对于沉默不产生可见表型的其他RNAi靶标特别有用。使用人工GFP报告基因的一个局限性是，外源性和内源性序列在跨代RNAi11中受到差异调控。因此，应考虑将内源性靶标的研究，例如温度敏感的胚胎致死等位基因oma-1（zu405）1,11,16,25，作为荧光转基因报告基因的补充方法。
在 RNAi 遗传检测中对 ChIP 进行分析时，需要对处理和重复进行比较。首先，为了考虑样品之间起始材料的差异，将 ChIP 信号归一化为与“输入百分比”相同位点的输入信号。并行处理组蛋白 H3 ChIP 将有助于确定任何组蛋白修饰改变是否与核小体密度的变化相对应。此外，由于 ChIP 是一个多步骤过程，因此效率可能因样品而异。选择合适的阳性和阴性对照位点有助于评估和比较样品和实验之间的信噪比。此外，为了便于样品之间的比较，RNAi 靶标的 ChIP DNA qPCR 阈值循环值通常归一化为对照位点3、11、15、23、30、31。为了评估 RNAi 处理的效果，还比较了处理 RNAi 与对照或无 RNAi 条件下的 ChIP 信号比率。目前方法的一个局限性是，ChIP是用整只动物进行的，而对RNAi处理的反应可能是种系所特有的。克服这一警告的一种方法是使用分离的种系细胞核进行 ChIP。优化 ChIP 的其他技术考虑因素也在其他地方进行了广泛讨论32,33。
总体而言，该 RNAi 遗传和 ChIP 方案提供了一个详细且易于适应的基础，可以与其他技术相结合，以进一步探索跨代表观遗传调控。例如，可以从 ChIP DNA （ChIP-seq） 构建高通量测序文库，以更详细地了解 RNAi 靶标近端和全基因组尺度的染色质景观。

表观遗传允许基因调控信息在几代人之间传递，因此可以让父母的环境或经历影响他们的后代。在秀丽隐杆线虫中，由外源双链 RNA （dsRNA） 引发的种系基因沉默可以在未暴露于原始触发因素的后代中遗传多代 1,2,3,4。这一过程称为 RNA 干扰 （RNAi） 遗传，是秀丽隐杆线虫中几种相关的表观遗传沉默现象之一，包括 piRNA 引发的多代沉默 2,5、副突变/RNAe（RNA 诱导的表观遗传沉默）6,7,8 和多拷贝阵列引发的沉默 9，它们对小 RNA 和染色质调控机制有重叠但不同的要求.在秀丽隐杆线虫外源 RNAi 中，dsRNA 被加工成小干扰 RNA （siRNA），这些 RNA 与初级 Argonaute 蛋白复合物一起作用以识别其靶标 mRNA。这种靶向导致与次级 Argonautes 结合的次级 siRNA 扩增，从而通过细胞质和核沉默途径沉默靶向 mRNA。对于种系表达的 RNAi 靶标，核二级 Argonaute HRDE-1 和其他核 RNAi 因子靶向新生转录本，导致转录抑制和组蛋白甲基转移酶募集以沉积抑制性染色质标记，包括 H3K9me310。组蛋白 H3K9me3 通过 RNAi 遗传促进种系表达的绿色荧光蛋白 （GFP） 转基因的可遗传沉默的建立11,12。
该方案的目的是使用在种系中表达GFP-组蛋白融合蛋白的转基因作为RNAi遗传的报告基因，并使用染色质免疫沉淀和定量聚合酶链反应（ChIP-qPCR）测定报告基因位点组蛋白修饰的变化。该协议描述了一种标准化的基于平板的RNAi补料方法，以启动报告基因沉默。它还提供了通过碱性次氯酸盐处理（“漂白”）在子宫内胚胎与妊娠成虫分离来代代传代动物的详细时间表。还描述了通过荧光显微镜监测群体亚群中GFP沉默频率的方法和代表性数据以及组蛋白H3K9me3 ChIP-qPCR。基于报告基因的RNAi遗传测定提供了一个高度易处理的系统，从功能上剖析遗传和环境因素在表观遗传调控中的作用13,14，并且使用这种报告基因的遗传筛选已经鉴定了2,3,15所需的基因和负调控基因16,17跨代表观遗传的持续时间。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Bioshop</td>
			<td>AGA002</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Bioshop</td>
			<td>AMP201</td>
			<td>Make a 100 mg/mL solution in ultrapure water. Filter-sterilize and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Anti-H3K9me3 Rabbit Polyclonal Antibody</td>
			<td>Abcam</td>
			<td>ab8898</td>
			<td>Concentration is batch-dependent (0.9 - 1 mg/mL).</td>
		</tr>
		<tr>
			<td>Anti-Histone H3 Rabbit Polyclonal Antibody</td>
			<td>Abcam</td>
			<td>ab1791</td>
			<td>Concentration is batch-dependent (0.7 - 1 mg/mL).</td>
		</tr>
		<tr>
			<td>Bleach (6% Sodium hypochlorite)</td>
			<td>Lavo</td>
			<td>02358107</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans strain with GFP RNAi Reporter</td>
			<td>NA</td>
			<td>SX1263</td>
			<td>Sapetschnig et al. 2015 (ref. 7). A gift from E. Miska lab, University of Cambridge.</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>BioShop</td>
			<td>CAR544</td>
			<td>Make a 25 mg/mL solution in ultrapure water. Filter-sterilize and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Dynabeads Protein G Magnetic Beads</td>
			<td>Invitrogen</td>
			<td>10003D</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli strain HT115(DE3)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>HT115(DE3)</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli strain OP50-1</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>OP50-1</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M, pH 8.0)</td>
			<td>Invitrogen</td>
			<td>15575020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Stereoscope</td>
			<td>Zeiss</td>
			<td>Axio Zoom.V16</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde (37%)</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>50046</td>
			<td>Make a 1.25 M solution and store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>HEPES-KOH (1 M, pH 7.5)</td>
			<td>Teknova</td>
			<td>H1035</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Printed Slides, 10 wells</td>
			<td>VWR</td>
			<td>100488-904</td>
			<td></td>
		</tr>
		<tr>
			<td>IGEPAL CA-630 (Octylphenol ethoxylate)</td>
			<td>BioShop</td>
			<td>NON999</td>
			<td>Make a 10% (v/v) solution in ultrapure water and store at room temperature.</td>
		</tr>
		<tr>
			<td>IPTG (Isopropyl-&#946;-D-thiogalactoside)</td>
			<td>BioShop</td>
			<td>IPT001</td>
			<td>Make a 0.2 g/mL solution in ultrapure water. Filter-sterilize and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>1725122</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar Plates supplemented with 100 &#181;g/mL Ampicillin</td>
			<td>NA</td>
			<td>NA</td>
			<td>Standard lab recipe.</td>
		</tr>
		<tr>
			<td>Levamisole (Tetramisole hydrochloride)</td>
			<td>Sigma</td>
			<td>L9756</td>
			<td>Make a 200 mM solution in ultrapure water. Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>LiCl (8 M)</td>
			<td>Sigma</td>
			<td>L7026</td>
			<td></td>
		</tr>
		<tr>
			<td>M9 Buffer</td>
			<td>NA</td>
			<td>NA</td>
			<td>22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, 1 mM MgSO4.</td>
		</tr>
		<tr>
			<td>Magnetic Separator (1.5 mL tubes)</td>
			<td>Applied Biosystems</td>
			<td>A13346</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Separator (0.2 mL tubes)</td>
			<td>Permagen</td>
			<td>MSR812</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td>12541B</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slide</td>
			<td>Technologist Choice</td>
			<td>LAB-037</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl (5 M)</td>
			<td>Promega</td>
			<td>V4221</td>
			<td>For ChIP buffers.</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td>Make a 10 M solution and store at room temperature.</td>
		</tr>
		<tr>
			<td>NGM Plates</td>
			<td>NA</td>
			<td>NA</td>
			<td>1.7% (w/v) agar, 0.3% (w/v) NaCl, 0.25% (w/v) peptone, 1 mM CaCl2, 5 &#956;g/mL cholesterol, 25 mM Potassium phosphate pH 6.0, 1 mM MgSO4, 50 &#181;g/mL streptomycin.</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG (1 mg/mL)</td>
			<td>Cell Signaling Technology</td>
			<td>2729</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes (35 mm x 10 mm)</td>
			<td>Sarstedt</td>
			<td>82.1135.500</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10X)</td>
			<td>Fisher BioReagents</td>
			<td>BP3991</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid - Control RNAi&#160;</td>
			<td>Addgene</td>
			<td>L4440 (Plasmid #1654)</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid - GFP-targetting RNAi&#160;</td>
			<td>Addgene</td>
			<td>L4417 (Plasmid #1649)</td>
			<td>Note, alternative L4440-derived plasmids targeting GFP can be used.</td>
		</tr>
		<tr>
			<td>Primer pair [-3.3 kb upstream of gfp]</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td>F: AAACCAAAGGACGAGAGATTCA, R: GGCTCGATCAAGTAAAATTTCG</td>
		</tr>
		<tr>
			<td>Primer pair [+1.3 kb downstream of gfp]</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td>F: TCGACCAGTTCTAAAGTCACCG, R: ACGTGCGGGATCATTTCTTACT</td>
		</tr>
		<tr>
			<td>Primer pair [clec-18]</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td>F: TGCTCCATGACCTCAACAACA, R: AGTACAGTTCACCGATCCAGA</td>
		</tr>
		<tr>
			<td>Primer pair [gfp exon 1]</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td>F: CTGGAGTTGTCCCAATTCTTGT, R: GGGTAAGTTTTCCGTATGTTGC</td>
		</tr>
		<tr>
			<td>Primer pair [gfp exon 4]</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td>F: GATGGCCCTGTCCTTTTACCA, R: ATGCCATGTGTAATCCCAGCA</td>
		</tr>
		<tr>
			<td>Primer pair [hrp-2]</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td>F: CGTCAACAGGGAGCAGCTG, R: CCTCCGAACTTTCTCTGTCCA</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablet</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K&#160;</td>
			<td>Bioline</td>
			<td>BIO-37084</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>CFX96&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAi-NGM plates</td>
			<td>NA</td>
			<td>NA</td>
			<td>1.7% (w/v) agar, 0.3% (w/v) NaCl, 0.25% (w/v) peptone, 1 mM CaCl2, 5 &#181;g/mL cholesterol, 25 mM Potassium phosphate buffer pH 6.0, 1 mM MgSO4, 25 &#181;g/mL carbenicillin and 5 mM IPTG.</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Sigma</td>
			<td>R4642</td>
			<td></td>
		</tr>
		<tr>
			<td>Sarkosyl (N-Lauroylsarcosine sodium salt)</td>
			<td>Sigma</td>
			<td>L5777</td>
			<td>Make a 10% (w/v) solution and store at room temperature protected from light for a maximum of 1 month.</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>74255</td>
			<td>Make a 10% (w/v) solution and store at room temperature.</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>30970</td>
			<td>Make a 5% (w/v) solution and store at room temperature protected from light for a maximum of 1 month.</td>
		</tr>
		<tr>
			<td>Sonication Tube</td>
			<td>Evergreen</td>
			<td>214-3721-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonication Tube Cap</td>
			<td>Evergreen</td>
			<td>300-2911-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Qsonica</td>
			<td>Q800R3-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Bioshop</td>
			<td>STP101</td>
			<td>Make a 50 mg/mL solution in ultrapure water. Filter-sterilize and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>TAE buffer (1X)</td>
			<td>NA</td>
			<td>NA</td>
			<td>40 mM Tris, 20 mM acetate, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>Tally counter clicker</td>
			<td>Uline</td>
			<td>H-7350</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>Bioshop</td>
			<td>TET701</td>
			<td>Make a 5 mg/mL solution in ethanol and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>05-400-205</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (1 M, pH 8.0)</td>
			<td>Invitrogen</td>
			<td>15568025</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td>Make a 10% (v/v) solution in ultrapure water and store at room temperature.</td>
		</tr>
	</tbody>
</table>

analysis of transgenerational epigenetic inheritance in c. elegans using a fluorescent reporter and chromatin immunoprecipitation (chip)

跨代表观遗传 （TEI） 允许通过非编码 RNA 和染色质修饰等因素在不改变基因组序列的情况下通过种系传递信息。线虫秀 丽隐 杆线虫的RNA干扰（RNAi）遗传现象是研究TEI的有效模型，它利用了该模式生物的短生命周期、自我繁殖和透明度。在RNAi遗传中，动物暴露于RNAi会导致基因沉默和靶位点染色质特征的改变，在没有初始触发因素的情况下持续多代。该方案描述了使用种系表达的核绿色荧光蛋白（GFP）报告基因分析 秀丽隐杆线虫 中的RNAi遗传。报告基因沉默是通过给动物喂食表达靶向GFP的双链RNA的细菌来启动的。在每一代中，动物被传代以保持同步发育，并通过显微镜确定报告基因沉默。在选定的世代中，收集群体并对其进行染色质免疫沉淀 （ChIP）-定量聚合酶链反应 （qPCR） 处理，以测量 GFP 报告基因座的组蛋白修饰富集。这种用于研究RNAi遗传的方案可以很容易地修改并与其他分析相结合，以进一步研究小RNA和染色质途径中的TEI因子。

Epigenetic inheritance allows the transmission of gene regulatory information across generations and can therefore allow the environment or experiences of parents to affect their progeny. In C. elegans, germline gene silencing initiated by exogenous double-stranded RNA (dsRNA) can be inherited for multiple generations in progeny not exposed to the original trigger1,2,3,4. This process, termed RNA interference (RNAi) inheritance, is one of several related epigenetic silencing phenomena in C. elegans, including piRNA-initiated multigenerational silencing2,5, paramutation/RNAe (RNA-induced epigenetic silencing)6,7,8, and multicopy array-initiated silencing9, which have overlapping but distinct requirements for small RNA and chromatin regulation machinery. In C. elegans exogenous RNAi, dsRNA is processed into small interfering RNAs (siRNAs) that act in a complex with primary Argonaute proteins to recognize their target mRNA. This targeting leads to the amplification of secondary siRNAs that associate with secondary Argonautes to silence target mRNA through both cytoplasmic and nuclear silencing pathways. For germline-expressed RNAi targets, the nuclear secondary Argonaute HRDE-1 and additional nuclear RNAi factors target nascent transcripts, resulting in transcriptional repression and recruitment of histone methyltransferases to deposit repressive chromatin marks, including H3K9me310. Histone H3K9me3 promotes the establishment of heritable silencing of germline-expressed green fluorescent protein (GFP) transgenes by RNAi inheritance11,12.
The goal of this protocol is to use a transgene expressing a GFP-histone fusion protein in the germline as a reporter for RNAi inheritance and to assay changes in histone modifications at the reporter transgene locus using chromatin immunoprecipitation and quantitative polymerase chain reaction (ChIP-qPCR). This protocol describes a standardized plate-based RNAi feeding approach to initiate reporter silencing. It also provides a detailed timeline for passaging animals between generations by isolating in utero embryos from gravid adults by alkaline hypochlorite treatment (&#39;bleaching&#39;). Methods and representative data for monitoring the frequency of GFP silencing in a subset of the population by fluorescence microscopy and for histone H3K9me3 ChIP-qPCR are also described. Reporter-based RNAi inheritance assays provide a highly tractable system to functionally dissect the roles of genetic and environmental factors in epigenetic regulation13,14, and genetic screens using such reporters have identified both genes that are required for2,3,15 and genes that negatively regulate16,17 the duration of transgenerational epigenetic inheritance.

作者聚焦：秀丽隐杆线虫的 RNAi 遗传和 ChIP 

基于荧光报告基因和染色质免疫沉淀法（ChIP）分析 秀丽隐杆线虫 的跨代表观遗传

Research in our lab examines how chromatin-associated proteins, histone modifications, and small RNA pathway factors work together in the context of development and transgenerational epigenetic inheritance. The RNA inheritance assay, using a GFP reporter, has become a very powerful tool. This protocol describes how to standardize the preparation, scoring, and passaging of worms, and the preparation of ChIP samples, which can help to generate reproducible results.One of the experimental challenges when studying germline-expressed transgenes is isolating the effects in the animal's germline. We're currently using whole animals for ChIP, but in the future we would like to develop assays to examine chromatin, specifically in the germline. Our laboratory is interested in histone methylation and proteins that recognize these marks.Going forward, we would like to investigate the interplay between these chromatin readers and small RNA pathways in the context of RNAi inheritance at transgenes and the regulation of endogenous sequences.

携带种系表达的GFP-组蛋白H2B [mex-5p：：gfp-h2b：：tbb-2 3'UTR]7 报告基因（图2A）的动物通过喂养暴露于GFP RNAi或对照RNAi，并按照方案和 图1中的说明进行传代。使用荧光解剖显微镜对每一代的群体样本手动评分种系中的核GFP信号。在用GFP RNAi处理的评分P0和F1动物中，转基因的沉默是完全渗透的（图2B）。在F2世代中，表现出GFP沉默遗传的群体比例约为50%。到F5代时，大多数种群没有表现出沉默的遗传，而到F10代时，没有检测到遗传，因为所有动物都表达了GFP。
为了确定与 RNAi 诱导的沉默相对应的组蛋白 H3K9me3 富集的变化，在 GFP RNAi 或对照 RNAi 处理后对 F1 代动物进行 ChIP-qPCR。正如预期的那样，与对照RNAi处理的动物相比，GFP RNAi处理的群体在GFP靶标和1.3 kb下游区域显示出更高的组蛋白H3K9me3水平（图2C）。组蛋白 H3K9me3 ChIP 的特异性得到了已知在该标记物中富集的阳性对照位点 （clec-18） 的富集的支持，但在附近的阴性对照位点 （hrp-2） 上没有。正如预期的那样，在所有qPCR位点中也检测到IgG对照免疫沉淀中的组蛋白H3富集和近背景富集。当报告基因处的 ChIP 富集归一化为 clec-18 阳性对照位点时，GFP RNAi 上的组蛋白 H3K9me3 富集更高，而组蛋白 H3 富集在对照组和 GFP RNAi 处理组之间相似（图 2D）。由于 GFP RNAi 预计不会影响组蛋白 H3K9me3 或 clec-18 位点的总组蛋白 H3 占有率，因此这种归一化可减轻技术差异，例如 GFP RNAi 和对照 RNAi 样品之间的 ChIP 效率差异。在 GFP RNAi 诱导的沉默后，组蛋白 H3K9me3 和组蛋白 H3 水平在 RNAi 处理之间的倍数变化显示 GFP 报告基因特异性组蛋白 H3K9me3 富集，与组蛋白占有率无关（图 2E）。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65285/65285fig02.jpg"> 图 2：GFP RNAi 诱导的沉默对应于 RNAi 靶标处 H3K9me3 富集升高。 （A） 种系表达的GFP RNAi报告基因mjIs134[mex-5p：：gfp-h2b：：tbb-2 3'UTR]的示意图，qPCR扩增子区域标记。（B） 在21°C下RNAi处理后跨代GFP表达评分。 误差线表示两个生物学重复的标准偏差。（C） 来自两个生物学重复的 F1 年轻人中 H3K9me3、组蛋白 H3 和 IgG 对照的 ChIP-qPCR。clec-18 和 hrp-2 分别是 H3K9me3 富集的阳性和阴性对照位点。（D） GFP RNAi 报告基因处的 H3K9me3 和组蛋白 H3 富集归一化为 clec-18 阳性对照位点。（E） GFP RNAi 和对照 RNAi 处理的动物之间 H3K9me3 和组蛋白 H3 富集的变化，归一化为 clec-18。虚线表示 1 的倍数变化。<a href="https://www.jove.com/files/ftp_upload/65285/65285fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

Watch this Scientific Journal Video about RNAi Inheritance and ChIP in C. elegans at JoVE.com

该方案描述了RNA干扰和ChIP测定，以研究 秀丽隐杆线虫中RNAi诱导的沉默和相关染色质修饰的表观遗传。

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through ...

我们实验室的研究检查了染色质相关蛋白、组蛋白修饰和小 RNA 通路因子如何在发育和跨代表观遗传的背景下协同工作。使用GFP报告基因的RNA遗传测定已成为一种非常强大的工具。该协议描述了如何标准化蠕虫的制备、评分和传代，以及 ChIP 样品的制备，这有助于产生可重复的结果。研究种系表达的转基因时，实验挑战之一是分离动物种系中的影响。我们目前正在使用整只动物进行ChIP，但未来我们希望开发检测方法来检查染色质，特别是在种系中。我们的实验室对组蛋白甲基化和识别这些标记的蛋白质感兴趣。展望未来，我们想在转基因的RNAi遗传和内源性序列的调控的背景下研究这些染色质读取器和小RNA通路之间的相互作用。

analysis-transgenerational-epigenetic-inheritance-c-elegans-using

Research

JoVE Journal

Genetics

Analysis of Transgenerational Epigenetic Inheritance in C. elegans Using a Fluorescent Reporter and Chromatin Immunoprecipitation (ChIP)

1.6K 次观看.  University of Toronto. 我们实验室的研究检查了染色质相关蛋白、组蛋白修饰和小 RNA 通路因子如何在发育和跨代表观遗传的背景下协同工作。使用GFP报告基因的RNA遗传测定已成为一种非常强大的工具。该协议描述了如何标准化蠕虫的制备、评分和传代，以及 ChIP 样品的制备，这有助于产生可重复的结果。研究种系表达的转基因时，实验挑战之一是分离动物种系中的影响。我们目前正在使用?...

视频: 作者聚焦：秀丽隐杆线虫的 RNAi 遗传和 ChIP 

Transgenerational epigenetic inheritance (TEI) allows the transmission of information through the germline without changing the genome sequence, through factors such as non-coding RNAs and chromatin modifications. The phenomenon of RNA interference (RNAi) inheritance in the nematode Caenorhabditis elegans is an effective model to investigate TEI that takes advantage of this model organism's short life cycle, self-propagation, and transparency. In RNAi inheritance, exposure of animals to RNAi leads to gene silencing and altered chromatin signatures at the target locus that persist for multiple generations in the absence of the initial trigger. This protocol describes the analysis of RNAi inheritance in C. elegans using a germline-expressed nuclear green fluorescent protein (GFP) reporter. Reporter silencing is initiated by feeding the animals bacteria expressing double-stranded RNA targeting GFP. At each generation, animals are passaged to maintain synchronized development, and reporter gene silencing is determined by microscopy. At select generations, populations are collected and processed for chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR) to measure histone modification enrichment at the GFP reporter locus. This protocol for studying RNAi inheritance can be easily modified and combined with other analyses to further investigate TEI factors in small RNA and chromatin pathways.

This protocol describes an RNA interference and ChIP assay to study the epigenetic inheritance of RNAi-induced silencing and associated chromatin modifications in C. elegans.

科研

遗传学

Bleaching and Plating Caenorhabditis elegans P0 Generation Embryos onto RNAi-NGM plates

Bleaching and Plating Caenorhabditis elegans P0 Generation Embryos onto RNAi-NGM plates  

Prepare animals for the RNAi inheritance assay by picking three or four gravid Caenorhabditis elegans adults under the 35 millimeter standard NGM plates seeded with OP50-1 bacteria. After four days, wash the gravid adult worms off the plates into 1.5 milliliter tubes using 800 microliters of M9 buffer supplemented with Triton X-100. Repeat the wash and pull the worms into one tube.After centrifuging the worms at 1, 000 G for 1.5 to two minutes, aspirate the supernatant leaving 100 microliters without disturbing the worm pellet. To each tube containing the worm pellet, add 650 microliters of double distilled water. To isolate the C.elegans embryos from the collected population, add 250 microliters of the freshly prepared bleaching solution and start a timer.Every one to two minutes, vortex the tubes thoroughly. After five minutes, monitor the degradation of adult worms under the stereoscope. Continue vortexing until the worms are completely dissolved and only embryos remain.This generally takes six to seven minutes. Immediately centrifuge the tubes at 1, 000 G for 1.5 minutes and aspirate the supernatant, leaving approximately 50 to 100 microliters of the supernatant with the embryos. Add one milliliter of M9 buffer supplemented with Triton X-100 to the embryos and vortex.Centrifuge the suspension and repeat the wash two times. After the final wash, aspirate the supernatant and leave approximately 100 microliters in the tube. Vortex the tubes with isolated embryos and pipette two microliters from each tube twice onto a labeled glass slide.Count the embryos using a tally counter to estimate the concentration of embryos per microliter. To start a semi-synchronized population growth, transfer approximately 250 embryos onto each RNAi NGM plate containing E.coli HT115 bacteria with GFP or control RNAi vectors. Allow the liquid to absorb.Incubate the P naught generation treated with RNAi for four days at 21 degrees Celsius.

漂白和接种 秀丽隐杆线 虫P0代胚胎到RNAi-NGM平板上

Passaging and Scoring the Caenorhabditis elegans P0 Generation for Germline GFP Expression

Passaging and Scoring the Caenorhabditis elegans P0 Generation for Germline GFP Expression  

Begin preparing the agarose pads by placing a drop of melted 1%agarose onto the vinyl record using a glass Pasteur pipet. Orient a glass slide so that the lines on the record are horizontal or vertical, and place the slide quickly onto the agarose drop for 30 seconds. Remove the glass slide from the record and add approximately five microliters of freshly prepared five millimolar levamisole under the stereoscope.Gently flick the tube containing worms and transfer 5 to 10 microliters onto the agarose pad to mount the worms. Estimate the number of worms as they are being added so that there are approximately 40 worms per replicate. Line up the worms in rows using an eyelash pick, and place a glass coverslip onto the slide.Isolate the embryos from the remaining worms using the bleaching protocol and plate 250 embryos onto 35 millimeter NGM plates seeded with OP50-1 bacteria. Under the fluorescent stereoscope, use a GFP filter set and count and record the number of GFP positive and GFP negative worms on the slides for each replicate using a tally counter. The manual scoring of the nuclear GFP signal in the germline for each generation showed that the silencing of the transgene was fully penetrant in the scored P0 and F1 worms treated with GFP RNAi.In the F2 generation, the proportion of the population exhibiting inheritance of GFP silencing was approximately 50%By the F5 generation, the majority of the population did not show the inheritance of silencing. By the F10 generation, all the worms expressed GFP and no inheritance was detected.

秀丽隐杆线虫 P0 代种系 GFP 表达的传代和评分

Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (ChIP)

Collection of F1 Generation Caenorhabditis elegans for Chromatin Immunoprecipitation (ChIP)  

Begin by plating approximately 3, 500 C.elgans F1 generation embryos on 14 plates and allow them to grow to the young adult stage for three days at 21 degrees Celsius. Wash and collect the C.elgans into 1.5-milliliter tubes using PBS/TX. Centrifuge at 1000 G for two minutes, aspirate, and pull the worms from one strain into one tube.Repeat three washes with PBS/TX. Next, aspirate the supernatant leaving one milliliter in the tube and invert to mix. After counting the number of C.elgans worms in three microliters three times, calculate the concentration of worms and transfer a volume corresponding to 3000 worms to a new tube for formaldehyde cross-linking.

用于染色质免疫沉淀 （ChIP） 的 F1 代 秀丽隐杆线 虫的收集

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP

Formaldehyde Crosslinking of the F1 Generation of Caenorhabditis elegans for ChIP  

Begin by adding formaldehyde to a 1.8%final concentration to the tube containing the worms and rotating the tube at room temperature for six minutes. Then immediately freeze the tubes in liquid nitrogen, and store the samples at 80 degrees Celsius. To proceed thaw the formaldehyde cross-link sample in a room temperature water bath for three minutes, and rotate the samples for 16 minutes at room temperature.At 1.25 molar glycine to a 125 millimolar final concentration, and rotate for five minutes at room temperature. Following this step, keep the samples on ice, or at four degrees, and use ice cold buffers until the magnetic bead elution. After centrifuging the sample at 1000 g for three minutes, wash it three times with one milliliter of PBSTX.Then wash it two times with one milliliter of Resuspension Buffer. After the last wash, leave enough Buffer to ensure the total volume is at least three times the pellet volume and a minimum of 100 microliters.

F1 代 秀丽隐杆线 虫的甲醛交联用于 ChIP

Sonication of Formaldehyde-Crosslinked Caenorhabditis elegans and Chromatin Immunoprecipitation

Sonication of Formaldehyde-Crosslinked Caenorhabditis elegans and Chromatin Immunoprecipitation  

Begin by mixing and aliquoting 90 to 120 microliters of the formaldehyde crosslinked C.Elegans samples into a polystyrene sonication tube. Add an equal volume of resuspension buffer containing 2X detergents. Sonicate in a water bath sonicator for seven minutes.Gently mix the sample by pipetting and repeat the sonication for an additional seven minutes. Once done, transfer the sonicated lysate to a 1.5 milliliter tube. Add a half volume of resuspension buffer without detergents.After centrifugation at 13, 000 G for 15 minutes at four degrees Celsius, divide the lysate supernatant into four parts. Store the input lysate part at minus 20 degrees Celsius in a 1.5 milliliter tube. Add 0.5 micrograms of anti-H3K9 trimethylation, anti-histone H3 or IgG to the appropriate immunoprecipitation or IP sample.Incubate the reaction at four degrees Celsius overnight with rotation. The following day, aliquot the nine microliters of protein G coated magnetic beads per IP sample in a 1.5 milliliter tube. After two washes with one milliliter of FA-150, resuspend the magnetic beads in the FA-150 buffer.Once done, add 7.5 microliters of the magnetic bead suspension to each IP antibody mix before incubating the tubes at four degrees Celsius for two hours with rotation. Next, wash the magnetic beads seven times using at least 200 microliters of each buffer solution. Incubate each wash at four degrees Celsius for five minutes with rotation before collecting the beads on a magnetic stand to aspirate the wash.After aspirating the last wash of the buffer, resuspend the magnetic beads in 50 microliters of chromatin IP elution buffer, and transfer it to a 1.5 milliliter tube. After eluting the sample in a ThermoMixer, collect the beads on a magnetic stand and transfer the supernatant to a new tube. Repeat the elution with another 50 microliters of chromatin immunoprecipitation elution buffer.Once done, pull a total of 100 microliters of supernatant before proceeding with reverse cross-linking and DNA elution.