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

Kirill Ustyantsev,Igor Sukhikh,Eugene Berezikov,Jakub Wudarski,Stijn Mouton,Filipa Reinoite,V Reinke

doi:10.1093/genetics/iyab076

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.

Highlights

Macrostomum lignano is a free-living flatworm that is gaining attention as a powerful model organism
The published transgenesis approach relies on random integration of DNA constructs into the genome
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

Summary

Introduction

Macrostomum lignano is a free-living flatworm that is gaining attention as a powerful model organism. Transposons offer opportunities for forward genetics studies, including insertional mutagenesis and trapping and mapping of functional DNA regulatory elements such as promoters, enhancers, and poly-adenylation signals (Bonin and Mann 2004; Kawakami et al 2004; Boulin and Bessereau 2007; Rad et al 2010; Song et al 2012; Casandra et al 2018) In this proof of principle study, we report transposon-mediated integration of piggyBac-derived genetic constructs in M. lignano using both the original and the hyperactive versions of the piggyBac transposase. We demonstrate that this method results in stable single-copy insertions with a frequency that is acceptable for practical applications

Materials and methods

Results and discussion

Literature cited