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

Abstract

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 via 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.

Regional signals in the planarian body guide stem cell fate in the presence of DNA instability

“…1C,D). Additionally, we utilized different strategies to functionally downregulate SmedRad51 with double-stranded-RNA (dsRNA) incorporated by feeding (Rouhana et al, 2013) or microinjections specifically protein expression is strongly downregulated in the whole organism by Rad51(RNAi). SMED-RAD51 was detected with anti-human RAD51 antibody and alpha tubulin was used as an internal loading control.…”

“…A total of five microinjections over four weeks were initially administered over three consecutive days and then one every 10 days. Alternatively, in vitro synthesized dsRNA was also administered by feeding to functionally disrupt genes by RNAi as described (Rouhana et al, 2013). Briefly, 1 µg of the Smed-Rad51-specific dsRNA was mixed with 30 µl of planaria food and was fed 3× times per week for 30 days.…”

Girardia dorotocephala transcriptome sequence, assembly, and validation through characterization of piwi homologs and stem cell progeny markers

“…Double-stranded RNA synthesized by T7 RNA Polymerase in vitro transcription was annealed by successive 3-min incubations at 90 °C, 75 °C, 50 °C and room temperature, and were then stored at −20 °C or used in feedings. Planarians were fed twice a week in a solution containing 1.5 μg dsRNA per 10 μl of liver paste (2:1 liver purée: water with food dye) as previously described (Rouhana et al, 2013). For regeneration assays, planarians were amputated five days after the fourth feeding, whereas planarians in homeostasis were continually fed for a total of 4 weeks or until phenotypes were observed.…”

“…The development of research tools to study gene expression and function in planarians, which include genome (Nishimura et al, 2015; Robb et al, 2015, 2008) and transcriptome (Brandl et al, 2016; Ishizuka et al, 2007; Nishimura et al, 2012; Sanchez Alvarado et al, 2002; Sasidharan et al, 2013; Zayas et al, 2005) sequences, as well as protocols for whole-mount in situ hybridization (King and Newmark, 2013; Pearson et al, 2009; Umesono et al, 1997) and RNA-interference (RNAi) (Newmark et al, 2003; Orii et al, 2003; Rouhana et al, 2013; Sanchez Alvarado and Newmark, 1999), have revitalized the use planarians as a model for molecular studies of stem cell-driven developmental processes (reviewed by Elliott and Sanchez Alvarado (2013), Newmark and Sanchez Alvarado (2002), Reddien and Sanchez Alvarado (2004), Rink (2013), Salo et al (2009) and Shibata et al (2010)). Planarians have also been the subject of recent work in toxicology, as well as behavioral, ecological, and evolutionary biology research (Alvarez-Presas et al, 2008; Hagstrom et al, 2016, 2015; Hicks et al, 2006; Inoue et al, 2015; Levin et al, 2016; Lombardo et al, 2011; Majdi et al, 2014; Shomrat and Levin, 2013), broadening their use as a model beyond fields related to developmental biology.…”

PIWI homologs mediate Histone H4 mRNA localization to planarian chromatoid bodies

“…DsRNA feeding was performed as described in Rouhana et al (2013). Planarians were fed liver solution containing dsRNA (0.1 µg/µl).…”