Establishment of a transgenic protocol in Trichoplax adhaerens

Trichoplax adhaerens, one of the simplest multicellular organisms belonging to the phylum Placozoa, occupies a basal position within Metazoa and serves as a critical model for investigating early multicellular evolution. Despite its significance, the absence of functional genetic tools has limited research to comparative genomics, ultrastructural characterization, and behavioral observation. Developing methodologies such as transgenesis and gene editing would substantially expand experimental capabilities. However, the absence of sexual reproduction precludes the use of conventional approaches like single-cell microinjection. To overcome these limitations, the present study established a transgenic method for expressing exogenous genes in T. adhaerens. Systematic evaluation of multiple transfection techniques identified electroporation as the most effective strategy for inducing ectopic gene expression in this species. Electroporation parameters were optimized to maximize efficiency while minimizing cytotoxicity. Using this approach, robust expression of green fluorescent protein (GFP) was achieved using various promoters. Ectopic expression of the puromycin acetyltransferase ( PAC) gene conferred increased resistance to puromycin exposure, suggesting a potential strategy for generating stable transgenic lines in the future. This work introduces a reliable framework for ectopic gene expression in T. adhaerens, enabling molecular and functional analyses in one of the earliest-diverging metazoan lineages.