Exploration of Non-Viral Gene Delivery Methods for Safe and Efficient Protein Expression in Non-Dividing Mammalian Cell Types

Abstract

Lentiviral vectors are a ubiquitous gene transfer tool in fundamental biological research. Although highly efficient and cost effective under most use cases, the limitations of lentivirus become apparent in specialized circumstances, such as under the MOSAIC (Multiplexed Optical Sensors Arrayed in Islands of Cells) imaging scheme. In this study we explored three possible avenues for the development and application non-viral transfection techniques compatible with the MOSAIC scheme; plasmid-mediated genomic integration, cytoplasmic transcription, and surface-mediated transfection. We did not measure any significant difference in stable expression with or without co-transfection of lentiviral packaging plasmid psPAX2 alongside expression mRNA and linear DNA nucleic acid species, indicating that plasmid-mediated genomic integration may be impossible. Protein expression resulting from cytoplasmic transcription was poor, likely indicating that transcribed mRNA was unstable and degraded quickly before sufficient translation could take place. Surface-mediated transfection was achieved by use of amorphous calcium phosphate crystals formed on the cell culture surface. Efficiency however was poor compared to traditional calcium phosphate transfection, likely due to large crystal size. Ultimately, further study and optimization will be required before any of these methods can be fully applied to the MOSAIC scheme.