Proof of principle for piggyBac-mediated transgenesis in the flatworm Macrostomum lignano

Abstract

Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. The free-living flatworm Macrostomum lignano is currently the only flatworm where stable transgenesis is available, and as such it offers a powerful experimental platform to address questions that were previously difficult to answer. The published transgenesis approach relies on random integration of DNA constructs into the genome. Despite its efficiency, there is room and need for further improvement and diversification of transgenesis methods in M. lignano. Transposon-mediated transgenesis is an alternative approach, enabling easy mapping of the integration sites and the possibility of insertional mutagenesis studies. Here, we report for the first time that transposon-mediated transgenesis using piggyBac can be performed in M. lignano to create stable transgenic lines with single-copy transgene insertions.

Optimized protocols for RNA interference inMacrostomum lignano

“…2D). Tim29 was added as it represents a more 'challenging' phenotype which is only observed in more extreme conditions such as whole-body regeneration and is very variable between worms (MOUTON et al 2021). Besides the negative control that lacks dsRNA during desalting, we also added a single soak in regular f/2 with dsRNA.…”

“…The one feature setting it apart from other flatworm models is the availability of successful methods for transgenesis (W udarski et al . 2017; U styantsev et al . 2021).…”

“…In addition, M. lignano has significant experimental power thanks to the development of diverse molecular tools and resources. The one feature setting it apart from other flatworm models is the availability of successful methods for transgenesis (WUDARSKI et al 2017;USTYANTSEV et al 2021). In recent years, high-quality genome and transcriptome assemblies (WASIK et al 2015;GRUDNIEWSKA et al 2016;WUDARSKI et al 2017;GRUDNIEWSKA et al 2018), and the transcriptional profiling of the germline and somatic neoblasts have been published (GRUDNIEWSKA et al 2016).…”

“…To ensure a constant supply of dsRNA to the animals, RNAi studies have always been performed by daily replacing the solution of dsRNA in a culture medium, in which the worms and algae (their food) are maintained. This approach has resulted in the identification of knockdown phenotypes for multiple genes involved in different biological processes, including regeneration, gametogenesis, and adhesion (SEKII et al 2009;KUALES et al 2011;GRUDNIEWSKA et al 2016;GRUDNIEWSKA et al 2018;MOUTON et al 2021). However, this approach has some significant limitations.…”

A genetic and microscopy toolkit for live imaging of whole-body regeneration inMacrostomum lignano

“…In contrast to other commonly studied flatworm models like planarians, which have limited transgenic tools (Hall et al, 2022), M. lignano has the ability to integrate exogenous DNAs into its genome, enabling transgenesis (Wudarski et al, 2017; Ustyantsev et al, 2021; Wudarski et al, 2022), which is further facilitated by the animal’s short egg-to-egg generation time (3 weeks at 20 ℃) and abundant, injectable zygotes (Mouton et al, 2018). However, our repertoire of transgenic tools is in its infancy and advanced microscopy tools capable of imaging entire organisms in their native physiological state throughout regeneration have been lacking.…”