Pharmaceutics 2025, 17(7), 905; https://doi.org/10.3390/pharmaceutics17070905 (registering DOI) - 12 Jul 2025
Abstract
Background/Objectives: Co-transfection of multiple DNAs is important to many research and therapeutic applications. While the optimization of single plasmid transfection is common, multiple plasmid co-transfection analyses are limited. Here we provide empirical data regarding multiple plasmid co-transfection while altering the number of species
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Background/Objectives: Co-transfection of multiple DNAs is important to many research and therapeutic applications. While the optimization of single plasmid transfection is common, multiple plasmid co-transfection analyses are limited. Here we provide empirical data regarding multiple plasmid co-transfection while altering the number of species of plasmids transfected (up to four different plasmids) and the amount of plasmids/cell using the two most common non-viral techniques, electroporation and lipofection. Methods: A549 human lung epithelial cells were transfected using lipofectamine 2000 or electroporation with combinations of plasmids, each expressing one of four different fluorescent proteins from the CAGG promoter. Twenty-four hours later, cells were analyzed by spectral flow cytometry to determine the number of cells expressing each fluorescent protein and the amount of fluorescent signal of each protein in a cell. Results and Conclusions: For electroporation, while the fraction of cells expressing plasmids increased with increasing amounts of DNA, increasing the number of plasmid species did not alter the fraction of expressing cells and had no effect on levels of expression in individual cells. By contrast, for lipofection, the fraction of cells expressing plasmids was not affected by the amount of DNA added but both the fraction of cells expressing and the level of protein produced in these cells decreased for each plasmid species as the number of delivered species increased. For both lipofection and electroporation after single plasmid transfection, the expressing cells had greater numbers of plasmid copies/cell than non-expressing cells. Multiple plasmid lipofection resulted in more plasmid copies/cell in co-expressing than non-expressing cells. Multiple plasmid electroporation was the inverse of this with fewer plasmid copies/cell in co-expressing than non-expressing cells.
Full article
(This article belongs to the Special Issue Electroporation-Mediated Drug and Gene Delivery)
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