Abstract
Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. However, the lack of transgenesis methods considerably hampers their wider use. Here we report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. We demonstrate that microinjection of DNA constructs into fertilized one-cell stage eggs, followed by a low dose of irradiation, frequently results in random integration of the transgene in the genome and its stable transmission through the germline. To facilitate selection of promoter regions for transgenic reporters, we assembled and annotated the M. lignano genome, including genome-wide mapping of transcription start regions, and show its utility by generating multiple stable transgenic lines expressing fluorescent proteins under several tissue-specific promoters. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism.
Highlights
Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning
We reasoned that microinjection approaches used in other model organisms, such as Drosophila, zebrafish and mouse, should work in M. lignano eggs (Fig. 1d, Supplementary Movie 1)
We observed a similar 3–10% frequency of initial transgene expression, and only two instances of germline transmission, one of which resulted from the negative control experiment without co-injected meganuclease protein (Supplementary Fig. 2c). These results suggest that I-SceI meganuclease does not increase efficiency of transgenesis in M. lignano, but instead that exogenous DNA can be integrated in the genome by non-homologous recombination using the endogenous DNA repair machinery
Summary
Regeneration-capable flatworms are informative research models to study the mechanisms of stem cell regulation, regeneration, and tissue patterning. We report development of a transgenesis method for Macrostomum lignano, a basal flatworm with excellent regeneration capacity. The reported transgenesis method and annotated genome sequence will permit sophisticated genetic studies on stem cells and regeneration using M. lignano as a model organism. One essential technique still lacking in planarians; is transgenesis, which is required for in-depth studies involving e.g., gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing. We present a method for transgenesis in M. lignano using microinjection of DNA into single-cell stage embryos and demonstrate its robustness by generating multiple transgenic tissue-specific reporter lines. The developed transgenesis method, combined with the generated genomic resources, will enable new research avenues on stem cells and regeneration using M. lignano as a model organism, including in-depth studies of gene overexpression, dissection of gene regulatory elements, real-time imaging and lineage tracing
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