RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics.
RNA interference by feeding in vitro-synthesized double-stranded RNA to planarians: methodology and dynamics.
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DOI：
10.1002/dvdy.23950
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发表时间：
2013-06
影响因子：
2.5
通讯作者：
Newmark, Phillip A.
中科院分区：
文献类型：
--
作者：
Rouhana, Labib;Weiss, Jennifer A.;Forsthoefel, David J.;Lee, Hayoung;King, Ryan S.;Inoue, Takeshi;Shibata, Norito;Agata, Kiyokazu;Newmark, Phillip A.
评估基因功能的能力对于理解生物过程是必不可少的。目前，RNA干扰(RNAi)是唯一可用于评估浮游生物基因功能的技术，它是通过注射双链RNA(DsRNA)、浸泡或摄取表达dsRNA的细菌来诱导的。我们描述了一种简单而强大的RNAi方案，包括体外合成dsRNA，并将其喂给浮游生物。该方法的优点包括能够从任何载体中产生dsRNA而不需要亚克隆，解决了输入dsRNA的数量和质量上的歧义，以及时间和应用的简便性。我们已经使用这种方法详细地评估了在浮游生物中诱导RNAi的后勤工作，从dsRNA在肠道中的摄取和处理，到在新生母细胞、生殖系和胞体中敲除的时机和效率。我们还系统地比较了dsRNA递送的数量、频率和方式的影响。这种方法给出了稳健和可重复性的结果，并适合于高通量研究。总体而言，这种RNAi方法结合了目前可用于在浮游生物中传递dsRNA的方法的优点，提供了一个显著的进步，并有可能在其他系统中受益于RNAi方法。
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 via injection of double-stranded RNA (dsRNA), soaking, or ingestion of bacteria expressing dsRNA. 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. 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.
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RNAi屏幕揭示了肠道形态发生，分化和干细胞增殖的肠道调节因子。
影响因子：
11.8
作者：
Forsthoefel, David J.;James, Noelle P.;Escobar, David J.;Stary, Joel M.;Vieira, Ana P.;Waters, Forrest A.;Newmark, Phillip A.
通讯作者：
Newmark, Phillip A.
影响因子：
4.3
作者：
Gentile L;Cebrià F;Bartscherer K
通讯作者：
Bartscherer K
影响因子：
3.3
作者：
Barnes, WM;Rowlyk, KR
通讯作者：
Rowlyk, KR
影响因子：
--
作者：
BAGUNA, J
通讯作者：
BAGUNA, J
影响因子：
48
作者：
Calixto, Andrea;Chelur, Dattananda;Topalidou, Irini;Chen, Xiaoyin;Chalfie, Martin
通讯作者：
Chalfie, Martin