DoE-based optimization of transposon-based gene insertion using the Harbor-IN system

Efficient and safe genetic labeling of mammalian cells is essential for applications in cell tracking, functional assays, and high-throughput screening. In this study, we developed and optimized a biosafety level 1 (BSL-1) protocol for stable integration of fluorescent and luminescent reporter genes using a transposon-based system. A Design of Experiments (DoE) approach was employed to systematically optimize co-transfection conditions of plasmid DNA encoding the reporter gene and in vitro-transcribed RNA encoding the transposase. Four commonly used transfection reagents-Lipofectamine 3000, PEI, ViaFect, and jetPRIME-were evaluated for their efficiency and compatibility with the system. Key factors such as nucleic acid ratios, total cargo amount, and reagent concentration were varied to maximize integration efficiency while maintaining cell viability. The optimized protocol achieved robust and reproducible reporter expression across multiple cell types without requiring viral vectors or higher biosafety containment. This work provides a scalable, non-viral strategy for generating stably labeled cell populations, enabling downstream applications in imaging, functional genomics, and assay development under standard laboratory conditions.