Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression
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Polyethylenimine (PEI)-based transient gene expression (TGE) is nowadays a well-established methodology for rapid protein production in mammalian cells, but it has been used to a much lower extent in insect cell lines. A fast and robust TGE methodology for suspension Hi5 (Trichoplusia ni) cells is presented. Significant differences in size and morphology of DNA:PEI polyplexes were observed in the different incubation solutions tested. Moreover, minimal complexing time (< 1 min) between DNA and PEI in 150 mM NaCl solution provided the highest transfection efficiency. Nanoscopic characterization by means of cryo-EM revealed that DNA:PEI polyplexes up to 300–400 nm were the most efficient for transfection. TGE optimization was performed using eGFP as model protein by means of the combination of advanced statistical designs. A global optimal condition of 1.5 × 106 cell/mL, 2.1 μg/mL of DNA, and 9.3 μg/mL PEI was achieved through weighted-based optimization of transfection, production, and viability responses. Under these conditions, a 60% transfection and 0.8 μg/106 transfected cell·day specific productivity were achieved. The TGE protocol developed for Hi5 cells provides a promising baculovirus-free and worthwhile approach to produce a wide variety of recombinant proteins in a short period of time.
KeywordsHigh Five cells Polyethylenimine Transient gene expression Cryo-electron microscopy Dynamic light scattering Design of experiments
The authors would like to thank Dr. Paula Alves (Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal) for providing the BTI-TN-5B1-4 cell line and pITV5-eGFP plasmid vector. We also would like to thank Martí de Cabo and Mónica Roldán from Servei de Microscòpia of UAB for his support with the Cryo-EM and confocal microscopy, respectively. The help of Llorenç Badiella (Servei d’Estadística Aplicada, UAB) in developing the R code and on statistical analysis is also acknowledged. The help of José Amable Bernabé (Institut de Ciència de Materials de Barcelona, CSIC), Manuela Costa (Institut de Biotecnologia i Biomedicina, UAB), and Dr. Salvador Bartolomé (Departament de Bioquímica i de Biologia Molecular, UAB) for the assistance with DLS, cytometry, and fluorometry, respectively, are also appreciated. Eduard Puente-Massaguer is a recipient of a FPU grant from Ministerio de Educación, Cultura y Deporte of Spain (FPU15/03577). The research group is recognized as 2017 SGR 898 by Generalitat de Catalunya.
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Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants performed by any of the authors.
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