Name: rnai screening to identify postembryonic phenotypes in c. elegans
Uploaded: 2012-02-13T13:00:00
Description: Watch this Scientific Journal Video about RNAi Screening to Identify Postembryonic Phenotypes in C. elegans at JoVE.com

作者想感谢里克·帕吉特博士（瓦克斯曼研究所，罗格斯大学，新泽西州）DBL-1基因和注射标记，博士，克里Rongo（瓦克斯曼研究所，罗格斯大学，新泽西州）的礼物。巴特格兰特博士的实验室进行基因枪轰击低拷贝数一体化的GFP标记DBL-1构造。勒内·加西亚实验室提供在创造texIs100技术援助。勒内·加西亚，罗宾Lints，李洪敏秦实验室提供了富有成效的意见。这项工作的启动资金是由TAMHSC从分子和细胞医学系。由部门和医学院院长办公室TAMHSC学院提供的资金购买该化合物的范围和旋转盘共聚焦。

1。筛选菌株建设精心设计的筛选菌株屏幕上的成功是至关重要的，并已在别处3。对于一些研究人员，用应变表示有形的产品，从转基因的实验需要。许多窝藏集成转基因的菌株是从总部大楼或个别研究人员。如果转基因株所需的屏幕上，但不可用，那么它可以使用像轰击紫外/ TMP 5，4，或MOS座子插入6发表的方法产生。为了形象化我们感兴趣的蛋白质?...

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这里提出的RNAi筛选方法使胚后的表型正常（或基因）所需要的基因产物的敏感和快速分析。所示的例子是屏幕中的荧光标记的蛋白质的亚细胞定位有关的基因。然而，这项协议可以进行修改，以确定影响其他感兴趣的胚后表型的基因。 此方法采用候选基因的方法，通过使用RNA干扰库的优势。利用诱变技术正向遗传筛选确定随机引起的新的等位基因，这些等位基因需要大量时?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> NGM的琼脂</td><td>线虫的生长介质</td><td> IPM的科技，公司</td><td>可以准备NGM的琼脂协议25 </td></tr><tr><td> M9培养基</td><td> 22MM KH 2 PO 4计 ， 42MM的Na 2 HPO 4， 86mm的氯化钠， 1毫米硫酸镁4 </td><td></td><td> 26 </td></tr><tr><td>琼脂</td><td>电解二氧化锰化学品公司</td><td> 1.01614.1000 </td><td> NGM的板在水中的2％。 4％显微镜幻灯片垫（最初釜和微波炉融化后）水。 </td></tr><tr><td>细菌用胰蛋白胨</td><td>碧迪 - Difco公司的CP </td><td> 211677 </td><td> 0.25％ </TD&gt; </tr><tr><td>经IPTG </td><td>研究产品国际股份有限公司</td><td> I56000-5.0 </td><td> 1毫米的最终浓度</td></tr><tr><td>羧</td><td>研究产品国际股份有限公司</td><td> C46000-5.0 </td><td> 50μg/ ml的工作液</td></tr><tr><td> LB培养基伦诺克斯</td><td>碧迪 - Difco公司的CP </td><td> 240230 </td><td> 20克/升</td></tr><tr><td>四环素</td><td>西格玛</td><td> 268054 </td><td> 12.5μg/ ml的工作液</td></tr><tr><td>次氯酸钠</td><td>任何品牌</td><td> 5％的家用漂白水</td><td>使用新鲜的漂白剂。 </td></tr><tr><td>氢氧化钠</td><td>任何品牌</td><td>中科院1310-73-2 </td><td> 5 N股</td></tr><tr><td> M9培养基</td><td> wormlab配方簿</td><td> <a href="http://130.15.90.245/wormlab_recipe_book.htm#Commonlab">http://1</a>30.15.90.245/wormlab_recipe_book.htm＃Commonlab </td><td> 26 </td></tr><tr><td>左旋咪唑</td><td>西格玛</td><td> 31742 </td><td> 100微米 - 1毫米的工作稀释</td></tr><tr><td>叠氮化钠</td><td> Fisher Scientific则</td><td> S227好</td><td> 10毫米M9的工作稀释</td></tr><tr><td> 24孔板</td><td>格雷纳生物一</td><td> 662160 </td><td>厂商VWR分销商</td></tr><tr><td>显微镜载玻片</td><td>任何品牌</td><td> 75×25×1毫米</td><td></td></tr><tr><td>镜盖单</td><td>任何品牌</td><td> 22×22毫米No.1.5 </td><td>使用显微镜制造商建议的厚度。 </td></tr><tr><td>复式显微镜</td><td>卡尔蔡司公司</td><td> A1M </td><td>使用目标和过滤器，以满足实验的需要。 </td></tr><tr><td>媒体泵</td><td>稳压器Varistaltic泵</td><td>凯特 模型＃72-620-000 </td><td>使用油管和设置适当的机器</td></tr></tbody></table>

rnai screening to identify postembryonic phenotypes in c. elegans

线虫已被证明是一个有价值的发现和功能分析很多基因和基因途径1模型系统。在这个系统中的研究更先进的工具和资源，促进具有更微妙的表型或角色的基因的不断发现。 在这里，我们提出了一个广义的协议，我们确定C.适应利用RNAi的利益胚后表型线虫基因。这个过程很容易地修改，以检测表型的选择，无论光或荧光光学显微镜解剖或复合。此场次协议利用生物体的有形资产和C.分子工具线虫研究社会生产。作为一个例子，我们展示了一个综合的转基因表达的RNAi屏幕荧光产品的使用，以确定这是正常的本地化所需的基因产品在后期幼虫和成虫。首先，我们用全长cDNA插入市售的RNA干扰基因库。这个库有助于减少候选基因产品通过RNAi多个考生的快速识别。第二，我们产生了一个综合的利益我们fluorecently标签蛋白基因的表达RNAi敏感的背景。第三，暴露孵出的动物RNA干扰，这个屏幕允许有一个重要的胚胎的作用，否则屏蔽后胚胎的作用，在调节蛋白基因的产品鉴定。最后，该屏幕采用单细胞分辨率配备一个复合显微镜。

Watch this Scientific Journal Video about RNAi Screening to Identify Postembryonic Phenotypes in C. elegans at JoVE.com

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

我们描述了致敏的方法来确定胚后调节蛋白表达和定位<em&gt; C。线虫</em&gt;使用RNAi技术为基础的基因组的屏幕和一个综合的功能，荧光标记的蛋白质基因的表达。

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 ...

Research

JoVE Journal

Biology

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

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 ...

科研

生物学

MISSION esiRNA for RNAi Screening in Mammalian Cells

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

Bacterial Delivery of RNAi Effectors: Transkingdom RNAi

细菌提供的RNAi效应：Transkingdom RNA干扰

RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory ...

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

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

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 ...

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

Quantitative and Automated High-throughput Genome-wide RNAi Screens in C. elegans

定量和自动化高通量的全基因组RNAi屏幕 C。线虫</em

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

创建定义的气体环境研究缺氧对<em&gt; C。线虫</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

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 ...

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

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

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 ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...