Nanoscale characterization coupled to multi-parametric optimization of Hi5 cell transient gene expression
- 47 Downloads
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.
Compliance with ethical standards
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.
- Chan LCL, Reid S (2016) Development of serum-free media for lepidopteran insect cell lines. Humana Press, New York, pp 161–196Google Scholar
- Montgomery DC (2012) Design and analysis of experiments, 5th edn. Wiley, ArizonaGoogle Scholar
- Mori K, Hamada H, Ogawa T, Ohmuro-matsuyama Y, Katsuda T, Yamaji H (2017) Efficient production of antibody Fab fragment by transient gene expression in insect cells. J Biosci Bioeng xx:3–8Google Scholar
- Osz-Papai J, Radu L, Abdulrahman W, Kolb-Cheynel I, Troffer-Charlier N, Birck C, Poterszman A (2015) Insect cells–baculovirus system for the production of difficult to express proteins. Humana Press, New York, pp 181–205Google Scholar
- Palomares LA, Realpe M, Ramírez OT (2015) An overview of cell culture engineering for the insect cell-baculovirus expression vector system (BEVS). Springer, Cham, pp 501–519Google Scholar
- Peixoto JL (1987) Hierarchical variable selection in polynomial regression models. Am Stat 41:311Google Scholar
- Shoja Z, Tagliamonte M, Jalilvand S, Mollaei-Kandelous Y, De stradis A, Tornesello ML, Buonaguro FM, Buonaguro L (2015) Formation of self-assembled triple-layered rotavirus-like particles (tlRLPs) by constitutive co-expression of VP2, VP6, and VP7 in stably transfected high-five insect cell lines. J Med Virol 87:102–111CrossRefGoogle Scholar