Abstract
The generation of stable clones for biomolecule production is a common but lengthy and labor-intensive process. For complex molecules, such as viruses or virus-like particles (VLPs), the timeline becomes even more cumbersome. Thus, in the early stages of development, transient production methods serve as a reasonable alternative to stable clone construction. In this work, an investigation of a polyethylenimine- (PEI-) based transfection method for the transient production of Chikungunya (Chik) VLPs, a vaccine candidate molecule, was undertaken. This effort focuses on tracking cell population responses during transfection, understanding how process changes affect these responses, and monitoring patterns in cell performance over the culture duration. Plasmid labeling and VLP staining were employed to comprehensively track cells via flow cytometry and to draw correlations between plasmid DNA (pDNA) uptake and the resulting VLP expression. The method detected high transfection efficiency (≥97%) in all samples tested and demonstrated the capability to track kinetics of plasmid-cell binding. With varied transfection cell concentrations, the pDNA binding kinetics are altered and saturation binding is observed in the lowest cell concentration sample tested in less than 3 hours of incubation. Interestingly, in all samples, the flow cytometry analysis of relative pDNA amount versus VLP expression staining showed that cells which contained fewer pDNA complexes resulted in the highest levels of VLP stain. Finally, to determine the potential breadth of our observations, we compared daily expression patterns of ChikVLP with a reporter, monomeric GFP molecule. The similarities detected suggest the interpretations presented here to likely be more broadly informative and applicable to PEI-based transient production of additional biological products as well.
Highlights
Transient transfections comprise the introduction of foreign nucleic acids into a host cell for the temporary expression of a gene of interest
Chemical methods involve the introduction of chemicals which form electrostatic interactions with nucleic acids and the anionic cell surface to result in endocytotic uptake of the nucleic acid/chemical complexes
A m242 monoclonal antibody [20, 21] specific to the ChikVLP was labeled with Alexa488 (ThermoFisher) for cell surface staining of live cells to determine virus-like particles (VLPs)-expressing cells and the relative expression level
Summary
Transient transfections comprise the introduction of foreign nucleic acids into a host cell for the temporary expression of a gene of interest. Biological methods involve the use of viruses to introduce genetic material, known as transduction; these are generally highly efficient, but disadvantages include lab hazards and mutagenesis [7] Physical methods include those that physically alter the cells to temporarily compromise the cell membrane for translocation of nucleic acids inside the cells. Physical-based methods comprise the most recent developments for transfection and include direct microinjection, biolistic particle delivery, electroporation, and laser-based transfection These methods are reported to be efficient but require specialized equipment [8]. Common chemical methods include the use of cationic polymers, calcium phosphate, cationic lipids, and cationic amino acids These methods are generally reported to be efficient, but may result in chemical toxicity to the cells [9]
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