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[Bio101] RNA Interference (RNAi) by Bacterial Feeding
[Bio101] 通过喂食细菌的RNA干扰 (RNAi) 方法   

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Abstract

There are 3 ways to perform RNAi in worms: microinjection, soaking and feeding. In the feeding protocol, RNAi is induced by cultivating worms on bacteria expressing gene-specific dsRNA. dsRNA is expressed in E. coli and ingested by worms. This protocol describes the feeding protocol to induce RNAi.

Materials and Reagents

  1. C. elegans RNAi clone/libraries (Open Biosystems or Source BioScience LifeSciences)
  2. Ampicillin (IBI Scientific)
  3. Tetracycline
  4. IPTG (Gold Biotechnology)
  5. LB agar medium: Agar (BD Biosciences), tryptone (BD Biosciences), yeast extract (BD Biosciences), NaCl (Research Organics)
  6. RNAi plate (NGM/IPTG/Ampicillin) (see Recipes)

Equipment

  1. Aluminum foil
  2. Petri dish (35 x 10 mm)

Procedure

  1. Streak dsRNA-expressing E coli onto LB agar plate containing ampicillin (50 μg ml-1) and tetracycline selection (12.5 μg ml-1) and incubate at 37 °C overnight.
    Note: There are two RNAi feeding libraries. One was constructed by the Vidal and Heuvel lab and can be ordered from Open Biosystems; the other was constructed by Julie Ahringer's lab and is available at Source BioScience LifeSciences.
  2. Inoculate bacteria in 3 ml LB liquid medium containing ampicillin (100 μg ml-1) only and incubate at 37 °C overnight.
  3. Spin down all 3 ml culture and pour off supernatant to 150 μl (concentrate culture by 20x). Resuspend pellet.
  4. Transfer 50 μl of cell resuspend to center of RNAi plate (NGM/IPTG/Ampicillin). Let dry (wrapped in aluminum foil) and induce overnight at room temperature (RNAi-seeded plates can be stored at RT for 2-3 days before use).
  5. Place 10-15 egg-laying worms on each plate. Incubate 2 - 6 h at 20 or 25 °C. Suck off parents and incubate at desired temperature until desired stage for further experiments.
    Note: Instead of egg lay, synchronize worms by bleach and transfer starved L1 larva to RNAi plates.

Recipes

  1. RNAi plate (NGM/IPTG/Ampicillin)
    Use the same recipe for making NGM agar medium but instead of adding streptomycin, add IPTG to final a concentration of 1 mM and ampicillin with a concentration of 100 μg ml-1. Typically pour onto small petri dish (35 x 10 mm).

References

  1. Timmons, L. and Fire, A. (1998). Specific interference by ingested dsRNA. Nature 395(6705): 854.
  2. Rual, J. F., Ceron, J., Koreth, J., Hao, T., Nicot, A. S., Hirozane-Kishikawa, T., Vandenhaute, J., Orkin, S. H., Hill, D. E., van den Heuvel, S. and Vidal, M. (2004). Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14(10B): 2162-2168.
  3. Kamath, R. S. and Ahringer, J. (2003). Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30(4): 313-321.

简介

有3种方法在蠕虫中进行RNAi:显微注射,浸泡和喂养。 在喂养方案中,通过在表达基因特异性dsRNA的细菌上培养蠕虫来诱导RNAi。 dsRNA在em中表达。 大肠杆菌并通过蠕虫摄入。 该协议描述诱导RNAi的喂养方案。

材料和试剂

  1. C。 elegans RNAi克隆/文库(Open Biosystems或Source BioScience LifeSciences)
  2. 氨苄青霉素(IBI Scientific)
  3. 四环素
  4. IPTG(黄金生物技术)
  5. LB琼脂培养基:琼脂(BD Biosciences),胰蛋白胨(BD Biosciences),酵母提取物(BD Biosciences),NaCl(研究机构)
  6. RNAi板(NGM/IPTG /氨苄青霉素)(参见配方)

设备

  1. 铝箔
  2. 培养皿(35×10mm)

程序

  1. 条纹表达dsRNA的大肠杆菌在含有氨苄青霉素(50μg/ml)和四环素选择(12.5μg/ml)的LB琼脂平板上,并在37℃下孵育过夜 注意:有两个RNAi饲料库。一个是由Vidal和Heuvel实验室构建的,并且可以从Open Biosystems订购;另一个由Julie Ahringer的实验室构建,并可在Source BioScience LifeSciences获得。
  2. 将细菌接种在含有氨苄青霉素(100μgml -1 -1)的3ml LB液体培养基中,并在37℃下孵育过夜。
  3. 旋转所有3毫升的文化和倒出上清液到150微升(浓缩培养20x)。重悬沉淀。
  4. 转移50微升细胞重悬到RNAi板(NGM/IPTG /氨苄青霉素)的中心。让干燥(包裹在铝箔中)并在室温下诱导过夜(使用前,RNAi接种的板可以在室温下储存2-3天)。
  5. 在每个板上放置10-15个产卵蠕虫。在20或25°C孵育2 - 6小时。抽出父母,在所需温度下孵育,直到进行进一步实验所需的阶段 注意:您不需要生蛋,就可以通过漂白剂,并将饥饿的L1幼虫转移至RNAi板。

食谱

  1. RNAi平板(NGM/IPTG /氨苄青霉素)
    使用相同的方法制作 NGM琼脂培养基,而不是添加 链霉素,加入IPTG至终浓度为1mM,加入浓度为100μg/ml的氨苄青霉素。 通常倒在小培养皿(35×10 mm)上

参考文献

  1. Timmons,L。和Fire,A。(1998)。 摄取dsRNA的特异性干扰 Nature 395(6705) ):854.
  2. Rual,JF,Ceron,J.,Koreth,J.,Hao,T.,Nicot,AS,Hirozane-Kishikawa,T.,Vandenhaute,J.,Orkin,SH,Hill,DE,van den Heuvel, Vidal,M。(2004)。 利用基于ORFeome的RNAi文库改善 Caenorhabditis elegans 表型映射。 Genome Res 14(10B):2162-2168。
  3. Kamath,R.S。和Ahringer,J。(2003)。 在秀丽隐杆线虫中进行全基因组RNAi筛选。 < em>方法 30(4):313-321
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:He, F. (2011). RNA Interference (RNAi) by Bacterial Feeding. Bio-protocol Bio101: e59. DOI: 10.21769/BioProtoc.59;
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