Name: large-scale gene knockdown in c. elegans using dsrna feeding libraries to generate robust loss-of-function phenotypes
Uploaded: 2013-09-25T19:00:00
Description: 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

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...

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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 ...

Research

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Biology

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

Large-Scale Screens of Metagenomic Libraries

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 ...

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Generation of Stable Transgenic C. elegans Using Microinjection

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

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

Large Scale Zebrafish-Based In vivo Small Molecule Screen

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

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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

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

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

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 ...

C. elegans Chemotaxis Assay

C. elegans Chemotaxis Assay

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

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

Antibody Staining in C. Elegans Using "Freeze-Cracking"

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

Methods to Assess Subcellular Compartments of Muscle in C. elegans

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 ...