RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: Methodology and dynamics

被引:151
作者
Rouhana, Labib [1 ,2 ]
Weiss, Jennifer A. [1 ,2 ]
Forsthoefel, David J. [1 ,2 ]
Lee, Hayoung [3 ]
King, Ryan S. [1 ,2 ]
Inoue, Takeshi [3 ]
Shibata, Norito [3 ]
Agata, Kiyokazu [3 ]
Newmark, Phillip A. [1 ,2 ]
机构
[1] Univ Illinois, Howard Hughes Med Inst, Urbana, IL USA
[2] Univ Illinois, Dept Cell & Dev Biol, Urbana, IL USA
[3] Kyoto Univ, Grad Sch Sci, Dept Biophys, Kyoto, Japan
基金
美国国家科学基金会;
关键词
RNA-interference; RNAi; siRNA; planarian; Schmidtea mediterranea; Dugesia japonica; GERM-CELL SPECIFICATION; PLURIPOTENT STEM-CELLS; GENE-EXPRESSION; SCHMIDTEA-MEDITERRANEA; TRANSCRIPTOME ANALYSIS; HYBRIDIZATION METHOD; DUGESIA-JAPONICA; EXCRETORY SYSTEM; REGENERATION; REGULATORS;
D O I
10.1002/dvdy.23950
中图分类号
R602 [外科病理学、解剖学]; R32 [人体形态学];
学科分类号
100101 ;
摘要
Background The ability to assess gene function is essential for understanding biological processes. Currently, RNA interference (RNAi) is the only technique available to assess gene function in planarians, in which it has been induced by means of injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. Results We describe a simple and robust RNAi protocol, involving in vitro synthesis of dsRNA that is fed to the planarians. Advantages of this protocol include the ability to produce dsRNA from any vector without subcloning, resolution of ambiguities in quantity and quality of input dsRNA, as well as time and ease of application. We have evaluated the logistics of inducing RNAi in planarians using this methodology in careful detail, from the ingestion and processing of dsRNA in the intestine, to timing and efficacy of knockdown in neoblasts, germline, and soma. We also present systematic comparisons of effects of amount, frequency, and mode of dsRNA delivery. Conclusions This method gives robust and reproducible results and is amenable to high-throughput studies. Overall, this RNAi methodology provides a significant advance by combining the strengths of current protocols available for dsRNA delivery in planarians and has the potential to benefit RNAi methods in other systems. Developmental Dynamics 242:718-730, 2013. (c) 2013 Wiley Periodicals, Inc.
引用
收藏
页码:718 / 730
页数:13
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