这项工作是由卫生部MH097163的国家机构的资金支持。某些菌株是由政府总部大楼，这是由研究基础设施计划的美国国立卫生研究院办公室（P40-OD010440）提供资助。

注意：该协议可以被用来确定在各种组织类型的所需的各种生物过程的基因。由于屏幕中一个必要的质量控制，动物被喂正反两方面的控制双链RNA表达的细菌，以证明在目标组织的RNAi效应。 其他已发表 ​​馈送协议生长的dsRNA表达菌中为50μg/ ml的氨苄青霉素或25微克/毫升羧苄青霉素8-10的存在。我们已经发现，在这些条件生长的细菌失去了双链RNA的质粒在高频率。事实上，我们发现，即使当细菌在非常高的浓度（高达2毫克/毫升）的氨苄青霉素的细菌的50％以上的过夜生长不再含有质粒。而生长在羧苄青霉素通常导致更高的保留质粒，25微克/毫升不足以防止质粒丢失。相反，我们发现，羧苄西林浓度被要求的500微克/毫升的增长，系统蒸发散在液体培养基和2毫克/毫升羧苄青霉素被要求阻止质粒亏损时，细菌生长在固体琼脂基质中阻止质粒丢失。 因为质粒是保留拦截成功的关键，只有羧苄青霉素时使用dsRNA的细菌培养在这个协议。我们已经仔细优化在此协议中描述的细菌的生长培养物中使用的羧苄青霉素的浓度，以确保细菌不含有质粒没有在这些培养物中生长。然而，作为质量控制措施，质粒丢失可以在几个步骤的协议来确定。为了确保有效的击倒，80％以上的细菌在每一个步骤中都必须包含双链RNA的质粒。如果细菌在协议的任何步骤的20％以上已经失去了双链RNA的质粒，检查所使用的羧苄青霉素的质量。羧苄青霉素应新鲜它被使用的当天制备。准备<html>新鲜的羧苄青霉素和重复文化。 1。如何确定质粒亏损<ol><li>除去细菌的一个小样本（如在每个步骤相关描述），并将其添加到450微升无菌水。 </li><li>用100微升这种稀释的样品用无菌水混合溶液稀释彻底之间建立进一步的1:10 1:100和1:1000系列稀释。 </li><li>传播100μl的每个稀释到LB和LB AMP板，并在37℃下过夜孵育这些板</li><li>第二天，识别LB平板，它包含100和500的细菌菌落之间。计数菌落对这个板块和相应的LB AMP板数（包含相同的稀释细菌）。 </li><li>确定从这些菌落数质粒丢失。菌不含有该质粒是用式1计算的，应该对有效的dsRNA击倒小于15％的百分比。 </li></ol><img alt="&lt;/ HTML">“等式1”的src =“/ files/ftp_upload/50693/50693eq1.jpg”/&gt; 2。做重复的库板 概述：dsRNA的库将从制造商（OpenBiosystems），为在96孔板中10566的细菌克隆到达。在-80℃冷冻储存立刻板块。原始库板将被复制和所有馈送屏幕将使用重复的库板为细菌的来源。这是明智的存储正本和副本的库板在单独的冰柜。对于建议便于搬运，重复10只，每天的库板。 <ol><li>从-80℃冰箱中取出的库板（S），取出铝箔封口，同时培养仍然处于冻结状态。更换库板的塑料盖，将板在一个水平面上，在室温下解冻（不超过30分钟）。 </li></ol> 注：我们注意到一个显著p在原始库板的每个孔中的细菌的ercentage不含有质粒。然而，因为库的细菌是一种珍贵的资源，不建议在原始文库培养物来测试质粒丢失。 <ol start="2"><li>使用多道移液器，加150微升库复制媒体的一个空的96孔平底重复的板的各孔中。 </li><li>通过上下吹打四到五倍使用多道移液器设置为100微升混合解冻库的细菌培养物。然后取出10微升每个混合培养，并转移到重复板的相应孔中。继续从库板转移的文化复制制版一定要改变每次传输的枪头。 </li><li>之后从图书馆板所有文化已经转移，覆盖原来的库板采用BRAYER辊，以确保彻底的封新胶粘薄膜的水井。重新装上塑料盖子上盘和将其直立在-80℃冷冻，直到培养物已再冷冻（至少30分钟）。然后将原来的库板回库储存箱。 </li><li>摇重复的库板的文化平8-10小时，在37℃（450转上microshaker，轨道3毫米），。 </li><li>确定在每个重复的库板的质粒丢失。我们发现，这种细菌在原始库板的每个孔中的高比例不含有双链RNA的质粒。这些细菌不利于生长在重复的文库是很重要的。为了防止它们的生长，高浓度的羧苄青霉素用于在库中复制媒体（3毫克/毫升）。使用这些条件的复制盘的培养后发现，90％以上的细菌含有双链RNA的质粒。从每个重复板的代表性培养井中取出10微升样品，并将其添加到450微升无菌水。使用这些稀释的样品，确定在每个重复文库质粒丢失按照步骤1.1-1.4板。如果观察到显著质粒损失（&gt;菌中复制的库的20％不含有质粒），用新鲜的羧苄青霉素重拍培养基中，并从步骤2.1重复协议。 </li><li>温育后，直接将一个新的箔粘合片在重复的板孔中，并放置在板的盖子移到该箔。将重复的库板直立在-80°C冰箱（至少30分钟），直到冰冻。一旦冻结，移动板，用于长期存储重复的库的存储箱。 </li></ol> 3。上Omniplates使得图书馆的临时副本 概述：要开始准备食物的dsRNA的屏幕，从重复的库板的细菌被转移到固体琼脂omniplates和过夜培养。为了防止丢失质粒在固体培养基上，omniplates含有羧苄青霉素在2毫克/毫升。可以使用一段在这些平皿上的细菌一个星期，并显示没有质粒的损失。我们并没有在生长在omniplates超过一周时间测试细菌质粒丢失。 <ol><li>从-80℃冰箱中取出重复的库板（S），然后取下铝箔封口，而文化仍然处于冻结状态。去除箔后，更换板的塑料盖，并设置重复的库板在一个水平面上，在室温下解冻（不超过30分钟）。 </li><li>消毒布克尔96针复制。一个干净的复制的标签应用蒸馏水冲洗，然后在每次使用前灭菌。消毒复制器，将其放入乙醇浴（3/4深在耐热盘），然后烧乙醇涂引脚关闭使用本生灯。使火焰熄灭，然后重复。 </li><li>将灭菌复制到解冻的重复板的孔中，并用它来轻轻搅动培养物。小体积的培养将坚持以复制的引脚。 </li> &lt;LI&gt;小心地从重复板的孔中删除复制，并把它（引脚向下）轻轻地omniplate的表面，小心不要刺破琼脂表面。保留复制的omniplate表面上3-4秒，这样从复制器的引脚细菌被转移到琼脂表面。 </li><li>取出复制，并允许细菌培养体积小（2-3微升）吸收到琼脂。在37℃下倒置15-18小时的omniplates这些omniplates可第二天早上接种到96孔的液体培养。另外，含过夜细菌菌落omniplates可以存储长达七天，在4°C。 </li><li>到副本传输额外的复制版，冲洗复制技巧用无菌水，并通过将复制器在乙醇和燃烧两次，和以前一样重复灭菌过程。一旦消毒，复制器可用于下一个重复的板块。 </li><lI&gt;在omniplates过夜培养后确定质粒丢失。隔夜增长后omniplates的细菌菌落将作为食物喂养屏幕的临时来源。重要的是，80％以上的细菌在每个菌落含有的dsRNA。细菌倾向于更快地失去质粒对固体琼脂培养基比液体培养。通过接种1ml的灭菌水中有少量从每个菌落的细菌和使用该稀释样本按照步骤1.1-1.4，以确定质粒损失确定质粒的损失，以omniplates两个有代表性的菌落。后市展望：超过90％的细菌各殖民地将包含双链RNA的质粒。如果观察到显著质粒损失（&gt;从细菌菌落的omniplate 20％不含有质粒），使用新鲜的羧苄青霉素翻拍omniplate媒体和步骤3.1重复的协议。 </li></ol> 4。对于饲养蠕虫准备细菌 Ø概述：细菌供给蠕虫利用从omniplates菌制备为1 ml液体培养基（在96孔格式）。这些液体培养物生长过夜，产生饱和nonlogarithmic细菌培养。过夜生长可确保所有培养物将包含相似浓度的细菌，因此，所有蠕虫将被送入食物的量相同。没有这个生长过程中的质粒丢失应得到遵守。 <ol><li>分配1毫升细菌生长培养基的成2毫升深孔96孔板中使用重复移液器和50毫升Combitip每个孔中。 </li><li>将无菌复制到包含生成的细菌菌落omniplates表面的步骤3.1-3.6作出肯定的是引脚与每个96细菌菌落接触。 </li><li>小心地从omniplate的表面移动复制到深井制版肯定的是，销不刮的深水井的两边，直到沉浸在生长介质。移动复制器慢慢在方形运动的深孔板中撞出细菌进入生长培养基的内壁以下。注意不要溅到和交叉污染水井。 </li><li> 对照孔：对于每一个96孔深孔培养板中加入的dsRNA表达细菌一口井，将作为阳性对照的预期敲除表型和阴性对照添加包含空dsRNA的载体（pL4440）细菌到另一个井。我们重新条纹控制dsRNA的表达每周一次细菌在含四环素（15微克/毫升）和羧苄青霉素（2毫克/毫升），以使细菌保持新鲜和保持质粒的LB平板上。 </li><li>用无菌接种环从控制板（约在复制传送的细菌数量相同）中删除单井隔离菌落，接种一空井在深孔培养板。记录已接种的孔的位置。另外，与细菌c中的循环一个被移动到含有250微升生长培养基的Eppendorf管中，涡旋混合，然后用于接种多口井。 </li><li>撼动深孔板中以650转上microshaker平坦的位置（轨道：3毫米），在37℃过夜。文化将在第二天早上饱和。无论如何，在这些文化中使用的羧苄青霉素的浓度应防止质粒丢失。 </li><li>确定在1毫升过夜培养质粒丢失。培养物将被直接用于饲料蠕虫的画面。重要的是，80％以上的细菌在各培养物含有双链RNA的质粒。确定质粒丢失两个有代表性的文化按照步骤1.1-1.4。后市展望：超过90％的细菌在每一种文化都包含双链RNA的质粒。在即使发展到饱和状态这些过夜培养我们从来没有观察到显著质粒丢失。但是，如果观察到显著质粒损失（&gt; 20％），用新鲜的羧苄青霉素和代表重拍生长培养基从步骤4.1吃的协议。培养物可以从原始omniplates只要omniplates少于1-2周龄的重新启动。 </li><li>一旦用于启动1毫升文化，到omniplates被存储在4℃下，直到屏幕完成并且可以用作食物来源，开始任何再试验培养物。 </li><li>培养过夜孵育后，用一个12通道多道移液器从深孔板取出150微升细菌培养，并喷射到进料板的表面。它不转让，蠕虫会挖洞，而不是吃的双链RNA表达菌中刺破琼脂的表面是很重要的。传输可以在一个时间（ 图1）来完成四口井。要做到这一点，将移液器吸头的多道移液器的每第三个通道（因为有每行只有4口井在送料盘）。后每组4口井被转移，变更为新的，无菌的枪头。每一个深96孔培养板，需要96个转移到8个12孔喂养板。 </li><li>商店接种板直立在黑暗中过夜使细菌能吸收到琼脂。 </li></ol> 5。生成的L1 C.被捕线虫幼虫 概述：L1-被捕幼虫用于启动对dsRNA的喂养板同步虫文化。通过漂白妊娠成人的人群隔离健康的卵子，然后使卵子孵出的S-完整的媒体没有食物产生这些幼虫。在没有食物的L1孵出幼虫会停滞发育，产生的L1幼虫的同步人口。在屏幕上，准备新鲜的L1-被捕幼虫每个星期，因为这些饥饿的动物生病随着时间的推移，必须丢弃。在一个双链RNA馈送菌株中使用的动物的遗传背景的选择可以根据应用而变化，对神经元的屏幕ERI-1;林-15B突变体被推荐。 <ol><li>挑选10-20 L4拉rvae含有OP50细菌草坪，等待6-7天，直到板六个独立NGM板包含许多新鲜产卵和妊娠成年人。 </li><li>加入3毫升水，以每块板的表面从所述板除去动物和鸡蛋和用戴手套的手指，从各板的表面撞出鸡蛋，细菌和动物下水。一定要覆盖板的各个领域，特别注意细菌的草坪。使用玻璃吸管每块板去除水中的细菌，蠕虫和鸡蛋，传输至一个无菌的50ml锥形管中。 </li><li>加入另外3毫升的水，以每块板的表面，上下吹打在表面以去除所有剩余的细菌，蠕虫和鸡蛋。此卷添加到50ml锥形管中。 </li><li>旋转的锥形管在1,500 rpm进行1分钟的台式离心机。蠕虫和蛋应沉淀到管的底部和细菌应保持暂停。小心吸所有，但4毫升从管，一定要避免沉淀的蠕虫和鸡蛋的液体。 </li><li>重悬沉淀蠕虫中残留的水，并用玻璃吸管转移到一个无菌的15毫升锥形管中。使体积至10ml无菌水。 </li><li>如之前的转速为1500 rpm离心1分钟。吸出上清液留下200μl水，以免打扰蠕虫/卵沉淀。 </li><li>加入10毫升的水，以锥形洗虫和虫卵和以前一样旋转，以去除额外的细菌。 </li></ol> 注：步骤5.8-5.11虫和虫卵被暴露在漂白液。漂白剂会破坏虫和虫卵留相对安然无恙。然而，如果鸡蛋被留在漂白溶液太长，它们也将被破坏。设置当漂白剂在步骤5.8添加了一个计时器可以肯定，鸡蛋不保持接触漂白超过10分钟。 <ol start="8"><li>删除所有，但200微升上清液，再次避免了沉淀，再加入10毫升漂白溶液和启动一个定时器。 </li><li>孵育3-4分钟蠕虫和鸡蛋的漂白粉溶液，偶尔颠倒混合。然后旋转以1,500 rpm持续45秒在台式离心机。寻找在管的底部上的沉淀物，它应该是更小和更紧凑的比原颗粒和可能采取的一个黄色的外观。 </li><li>吸出漂白剂溶液离开200μl的后面，以便不打扰颗粒。 </li><li>添加另一个10毫升新鲜漂白液和之前反转。当计时器显示的是10分钟，再旋转，吸出的漂白粉溶液，留下200微升的背后，迅速加入10毫升无菌水。检查在解剖显微镜下的解决方案。幼虫和许多成年人应该溶解在漂白液，留下大部分鸡蛋落后。 </li><li>如前旋转45秒，然后再次吸出上清液，以便不打扰颗粒。这种颗粒应包含主要的鸡蛋，将是非常宽松的。 </li><li>添加ANOT她10毫升的水，颠倒混合，旋转和吸留一点水后面。它以除去尽可能多的漂白剂溶液，作为可能是重要的。 </li><li>加入4 mL S-完整的媒体到锥形管，重悬卵并转移到一个无菌的100ml锥瓶中。将烧瓶置于20℃培养箱中在振荡器上设置移动刚刚足够快，以使烧瓶中的内容，以漩涡。这将提供足够的通气支持L1幼虫，当他们孵化。在15小时所有的鸡蛋都应该有孵化生产的L1被捕幼虫的同步人口。 </li></ol>屏幕中被捕的L1幼虫应作出新的每周。一旦第一批已经作出，后续的批次可以通过添加500被捕L1幼虫（由前一周的批），以每四个标准，接种6厘米板（含OP50 100μL过夜培养）的启动，并孵育20 °C为4天。第四天板应该包含很多鸡蛋和妊娠成人和可漂白准备新鲜鸡蛋更同步L1幼虫。 6。传输被捕L1幼虫喂养上板 概述：要执行的dsRNA屏幕，20-30 L1被捕幼虫转移到双链RNA喂养板。动物们喂食dsRNA的表达菌2-3天后早期击倒效果，如幼虫逮捕或致死将被观察到。取决于所进行的画面上，期望表型可能要么在原动物放置在馈送盘（P0动物），或在它们的后代（F1动物）被观察到。表型的演讲将取决于许多变量，包括通过其表达撞倒的基因和在目的基因需要在开发过程中的时间编码的蛋白质的perdurance。开始的dsRNA喂养文化，只有20-30动物应该之前旁听既P0和F1的表型imals排气的食物来源。 20-30动物很容易地转移到进料板，首先要确定L1的浓度被捕动物被捕L1文化。 <ol><li>含混合被捕L1幼虫分发动物的锥形瓶中。 </li><li>在4-5删除将10μl和地方滴上非种子选手6厘米NGM板落下板的中心确保所有的媒体从吸管中排出。 </li><li>使用移液管尖的端部传播蠕虫悬浮液的点，形成的液体的单个连续线横跨板的直径。 </li><li>使用解剖显微镜下，计数所有的动物开始于液体的线的一端，并继续向另一端之前的液体吸收到板和动物的分散。这可能需要的解剖范围不断重新定位可以肯定，没有动物被错过。 </li><li>为三个独立的10微升重复此等分和平均3号以确定每微升虫悬浮液的蠕虫的平均数目，并直接在锥形烧瓶中记录该号码。 </li></ol> 7。开始喂养屏幕 概述：为了开始所述dsRNA屏幕20-30被捕L1幼虫加到包含一个薄的dsRNA表达细菌草坪12孔馈送板的每个孔中。馈送板包含标准线虫生长培养基（NGM），并补充有500微克/毫升羧苄青霉素（以防止质粒损失）和1mM IPTG（诱导的双链RNA的表达）。动物被允许在20°C养活并发展了好几天两个典型的喂养方案动物已经过期3天进行的F1画面时执行P0屏幕和6-7天的时候。馈送的持续时间，但是，可以根据寻求在屏幕中的特定的表型变化。 <ol><li>使用L1被捕幼虫和无菌水的悬浮准备这样禄含2,000蠕虫/毫升。每个12孔送料盘将需要120微升这种蠕虫病毒悬液。调匀，以确保均匀分布的动物及动物转移到一个无菌的贮存器移液。 </li><li>使用多道移液器设置与技巧在每一个第三的位置（ 见图1）转让10微升的蠕虫病毒液直接滴进料板的菌苔。小心不要刺破琼脂的表面，如蠕虫会钻入琼脂，而不是吃的双链RNA表达的细菌。 </li><li>混音在水库蠕虫溶液（轻轻晃动了大约）每两行喂养板后，蠕虫迅速解决。继续配药蠕虫暂停，直到所有进料板有虫。检查几口井早就下的解剖范围，确保每口井是越来越20-30蠕虫。 </li><li>店内板直立在20℃培养箱加湿箱。第二天，后禾RM悬浮液已被吸收到板，翻转板，并返回到雪茄盒在20℃下对这些板块的动物可以在3天后（P0为表型）进行观察后再次6天（F1的表型）。 </li></ol> 注意：术后7天虫生长留在送料盘的细菌已经过测试，质粒丢失和近100％保留了双链RNA的质粒。

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作者宣称，他们有没有竞争的财务权益。

我们已经描述了使用市售的dsRNA喂养库在C中进行大规模的屏幕协议线虫 。我们提供复制的库，并有效地使用它的所有指令。我们表明，这种方法能够鉴定参与在动物的不同组织中的不同生物过程的基因。虽然我们在这里描述的表型是容易观察到（UNC，EGL，和DPY），该协议可以适于以搜索所需的几乎任何生物过程的基因。例如，我们已经用这种协议来确定所需的神经传递的蠕虫25?...

从历史上看，遗传筛选中C。线虫已经被治疗动物用化学诱变剂例如乙基甲磺酸酯向DNA中创建随机突变进行。后代诱变的动物或grandprogeny然后分离出具有所期望的异常表型。负责引起表型的突变，然后通过一个劳动密集的映射过程或通过测序突变体蠕虫的整个基因组鉴定。有许多优势，进行这样的化学诱变屏幕，因为它们创建了蠕虫的基因组内的永久损伤，可能导致获得性功能的功能丧失和两个表型，但该映射过程是缓慢和昂贵。 双链RNA干扰（dsRNAi）提供鉴定参与重要生物学过程的基因替代的方法。在该方法中，dsRNA的匹配的内源基因的编码序列导入该蠕虫。所述dsRNA 在体内处理，生成较小（21-22核苷酸）的RNA充当导游来查找和绑定匹配的内源mRNA，针对他们毁灭1。这个过程是比较快的，但是由于双链RNA靶向的mRNA而不是DNA，还原功能只表型都采用这种方法观察到。 导入的dsRNA导入动物可以通过直接注射的dsRNA 2来实现，由或喂养表达dsRNA的4只动物的细菌浸泡动物中的dsRNA 3。每一种分娩方式引起全身击倒效果的动物大部分细胞表达的SID-1，能够导入的dsRNA 5的跨膜通道。本来作为一个反向遗传学的方法拦截单个基因的一个表达的时间，两个进料库的发展现在允许大规模的遗传筛选。在市售的dsRNA库包含巴代表所有三任55％或87％cterial克隆线虫基因6,7。 不幸的是，大规模的dsRNAi喂养的屏幕往往会造成变量击倒效率8-10。而击倒通过RNAi的绝对水平是不容易测量的，在大型屏幕中观察到的减少击倒效率必须由逸出降解通过RNAi残余基因特异性的mRNA而引起。这，反过来，可能是从细菌中这些屏幕饲喂给动物的dsRNA质粒丧失的直接结果。支持这样的想法，动物喂食细菌的混合物，其中只有50％的细菌表达针对靶基因的dsRNA，显示显着减少（如果有的话）时相比，控制馈动物11拦截效果。执行dsRNAi屏幕为大多数基因在C.当基因敲除的程度成为一个关键问题线虫是haplosufficient，因此基因的表达必须由至少50％T降低Ø造成损失的功能表型12。 我们使用先前公布的喂养方案来执行大型屏幕和报告发现别人8-10相同的变量击倒效果。在寻找可能的原因，我们发现，近60％的喂养动物在屏幕的细菌失去了双链RNA的质粒。我们已经开发了一个库中的重复和筛选方案，大大减少或消除质粒的损失，大大提高击倒效率。该协议适用于C。进行大规模的屏线虫 ，可用于筛选市售的dsRNA的库中的任一个。使用此协议，我们发现，基本上100％的画面中喂养动物的细菌保留了双链RNA编码的质粒，造成强大的和高渗透剂功能丧失表型检测的所有组织。

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>96-well Falcon flat bottom plate </td>
 <td>Fisher Scientific</td>
 <td>353072</td>
 <td>sterile</td>
 </tr>
 <tr>
 <td>adhesive foil</td>
 <td>USA Scientific</td>
 <td>2923-0100</td>
 <td></td>
 </tr>
 <tr>
 <td>Nunc omniplate</td>
 <td>Fisher Scientific</td>
 <td>267060</td>
 <td></td>
 </tr>
 <tr>
 <td>2.0 ml deep 96-well polypropylene Masterblock</td>
 <td>USA Scientific</td>
 <td>5678-0285</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>Lids for deep well plates</td>
 <td>USA Scientific</td>
 <td>5665-6101</td>
 <td></td>
 </tr>
 <tr>
 <td>50 ml combitips</td>
 <td>USA Scientific</td>
 <td>4796-5000</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>Reservoir</td>
 <td>USA Scientific</td>
 <td>2977-8500</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>12-well plates</td>
 <td>USA Scientific</td>
 <td>CC7682-7512</td>
 <td>individually wrapped</td>
 </tr>
 <tr>
 <td>dsRNA feeding library</td>
 <td>OpenBiosystems</td>
 <td>RCE1181</td>
 <td>store -80 &deg;C</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Fisher Scientific</td>
 <td>BP1760-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetracycline</td>
 <td>Fisher Scientific</td>
 <td>BP912-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbenicillin</td>
 <td></td>
 <td></td>
 <td>allow 2-4 weeks for delivery <a href="http://www.biochemicaldirect.com" target="_blank">www.biochemicaldirect.com</a></td>
 </tr>
 <tr>
 <td>Bacteriolgical Agar Ultrapure grade A </td>
 <td>Affymetrix</td>
 <td>10906</td>
 <td>for worm plates</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Brayer roller</td>
 <td>USA Scientific</td>
 <td>9127-2940</td>
 <td></td>
 </tr>
 <tr>
 <td>Boekel 96-pin replicator</td>
 <td>Fisher Scientific</td>
 <td>05-450-9</td>
 <td>autoclavable</td>
 </tr>
 <tr>
 <td>microshaker</td>
 <td>Thomas Scientific</td>
 <td>1231A93</td>
 <td>3 mm orbit</td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (0.5-10 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-0510</td>
 <td></td>
 </tr>
 <tr>
 <td>12 channel multichannel pipet (30-300 &mu;l)</td>
 <td>USA Scientific</td>
 <td>7112-3300</td>
 <td></td>
 </tr>
 <tr>
 <td>Repeater Plus manual pipettor</td>
 <td>USA Scientific</td>
 <td>4026-0201</td>
 <td></td>
 </tr>
 </tbody>
 </table>

large-scale gene knockdown in c. elegans using dsrna feeding libraries to generate robust loss-of-function phenotypes

通过喂食虫细菌表达dsRNA的RNA干扰一直是一个有用的工具，以评估在C基因功能线虫 。虽然这种策略效果很好，当少数基因的有针对性的击倒，大规模饲养的屏幕显示出不同击倒效率，这限制了它们的效用。我们已经解构先前公布的RNAi敲低的协议，发现降低击倒的主要来源可以归因于双链RNA编码的质粒从输送到动物的细菌的损失。基于这些观察，我们已经开发出一种dsRNA的喂养方案，大大减少或消除质粒的损失，实现高效率，高通量击倒。我们表明，该协议将产生稳定，可重复敲C的下降线虫基因在多种组织类型，包括神经元，并且将允许在大型屏幕有效击倒。该协议使用市售的dsRNA喂养图书馆和描述要复制的库和执行的dsRNA屏幕所需的所有步骤。该协议不需要使用任何复杂的设备，并因此可以通过任何C下执行线虫实验室。

P0敲除表型将开始出现后饲养动物的dsRNA表达菌2-3天。一些早期的表型包括幼虫逮捕和杀伤力，并应在所有饲养井的2-3％进行观察。这些相同的表型也将在F1代动物中观察到。 F1鸡蛋是不能孵化的存在是另一种常见的F1表型，并在一起，这三个表型将在近10％的F1屏幕所有孔的观察。 我们使用这里描述的协议，以检查对表达于多种组织类型为P0和F1的表型的多个基因敲低效果。因...

Watch this Scientific Journal Video about Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes at JoVE.com

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

而喂养的dsRNA在C线虫是一种功能强大的工具来评估基因功能，为大规模饲养屏幕当前的协议导致变量击倒效率。我们描述了一种改进的协议用于进行大规模的RNAi馈送屏幕，导致基因表达的高度有效的和可再现的击倒。

在大规模的基因敲除<em&gt; C。线虫</em&gt;使用双链RNA料库产生失去功能的鲁棒表型

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

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Research

JoVE Journal

Biology

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

11.9K 次观看.  University of Massachusetts, Amherst. 而喂养的dsRNA在C线虫是一种功能强大的工具来评估基因功能，为大规模饲养屏幕当前的协议导致变量击倒效率。我们描述了一种改进的协议用于进行大规模的RNAi馈送屏幕，导致基因表达的高度有效的和可再现的击倒。 

视频: Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

Large-Scale Screens of Metagenomic Libraries

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation. Generating a streamlined procedure for creating and screening metagenomic libraries is therefore useful for efficient high-throughput investigations into the genetic and metabolic properties of uncultured microbial assemblages. Here, key protocols are presented on video, which we propose is the most useful format for accurately describing a long process that alternately depends on robotic instrumentation and (human) manual interventions. First, we employed robotics to spot library clones onto high-density macroarray membranes, each of which can contain duplicate colonies from twenty-four 384-well library plates. Automation is essential for this procedure not only for accuracy and speed, but also due to the miniaturization of scale required to fit the large number of library clones into highly dense spatial arrangements. Once generated, we next demonstrated how the macroarray membranes can be screened for genes of interest using modified versions of standard protocols for probe labeling, membrane hybridization, and signal detection. We complemented the visual demonstration of these procedures with detailed written descriptions of the steps involved and the materials required, all of which are available online alongside the video.

宏基因组库的大型屏幕

Metagenomic libraries archive large fragments of contiguous genomic sequences from microorganisms without requiring prior cultivation.  Generating a ...

科研

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

斑马鱼为基础的大型在体内小分子屏幕

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

测量在C.elegan的RAN肽毒性

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...