Optimizing multi-gene co-expression with large plasmids

doi:10.1038/s41598-026-69501-7

Optimizing multi-gene co-expression with large plasmids

2026-09-06

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DOI：10.1038/s41598-026-69501-7

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Large plasmids are often avoided in mammalian co-transfection due to the assumption that they transfect poorly, driving the use of multiple smaller plasmids. Here, we pair finite-state-projection modeling with flow cytometry experiments to compare one-, two-, and three-plasmid delivery of GFP/BFP/RFP. Estimated entry rates were size-independent from 4.9 to 16.4 kb, indicating that plasmid length is not the dominant barrier in this range. Our results suggest that using lipofectamine slightly increases co-transfection efficiency due to the ability of lipoplexes to shuttle multiple plasmids for sizes < 17 kb. Additionally, we show that contrary to current beliefs, putting all genes onto the same plasmid both increases the probability that a cell will express all genes of interest and results in a tighter correlation of gene expression levels compared to these multi-plasmid systems. Ultimately, these results suggest that in the context of lipofectamine 2000-mediated transfection of plasmids sized 4.9–16.4 kb transiently expressing fluorescent reporters into HEK293 cells, single larger plasmids encoding multiple genes outperform equivalent systems using multiple smaller plasmids. This benefit suggests that single-plasmid design strategies should be investigated more thoroughly as they could benefit a broad variety of applications such as viral-vector production.

Keywords:

Co-transfection efficiency

Plasmid size

Multi-gene expression

Lipofectamine transfection

Stochastic modeling

Multi-plasmid delivery

Optimizing multi-gene co-expression with large plasmids

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