RNAi筛选识别胚后的表型 C。线虫</em
Summary
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> C。线虫</em>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。
Abstract
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。
Protocol
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?…
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Name of the reagent | Company | Catalogue number | Comments |
| NGM Agar | Nematode growth medium | IPM Scientific, Inc | Can be prepared following NGM agar protocol25 |
| M9 Medium | 22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl, 1 mM MgSO4 |
26 | |
| Agar-Agar | EMD Chemicals Inc. | 1.01614.1000 | 2% in water for NGM plates. 4% in water for microscope slide pads (autoclave initially and microwave to melt thereafter). |
| Bacto Peptone | Becton Dickinson – Difco CP | 211677 | 0.25% |
| IPTG | Research Products International Corp. | I56000-5.0 | 1 mM final concentration |
| carbenicillin | Research Products International Corp. | C46000-5.0 | 50 μg/ml working dilution |
| LB Broth Lennox | Becton Dickinson – Difco CP | 240230 | 20 g/liter |
| tetracycline | Sigma | 268054 | 12.5 μg/ml working dilution |
| sodium hypochlorite | Any brand | 5% household bleach | Use fresh bleach. |
| sodium hydroxide | Any Brand | CAS 1310-73-2 | 5 N stock |
| M9 medium | Wormlab Recipe Book | http://130.15.90.245/wormlab_recipe_book.htm#Commonlab | 26 |
| levamisol | Sigma | 31742 | 100 μM – 1 mM working dilution |
| sodium azide | Fisher Scientific | S227 | 10 mM in M9 working dilution |
| 24-well plate | Greiner Bio-One | 662160 | VWR distributor |
| microscope slides | Any brand | 75 x 25 x 1 mm | |
| microscope cover slips | Any brand | 22 x 22 mm No.1.5 | Use the thickness recommended by the microscope manufacturer. |
| compound microscope | Carl Zeiss, Inc. | A1m | Use objectives and filters to match the needs of the experiment. |
| media pump | Manostat Varistaltic pump | Kate model #72-620-000 |
Use tubing and settings appropriate for the machine |
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Citar este artigo
Beifuss, K. K., Gumienny, T. L. RNAi Screening to Identify Postembryonic Phenotypes in C. elegans. J. Vis. Exp. (60), e3442, doi:10.3791/3442 (2012).