Which one of the thermal approaches (heating DNA or cells) enhances the gene expression in mammalian cells?

Abstract

Objectives: Heat treatment as a physical method could increase the cellular uptake of nucleic acids. In this study, the effects of heat shock were evaluated to enhance the transfection efficiency of three plasmid DNAs into HeLa and TC-1 cancerous, and HEK-293 T and Vero non-cancerous cell lines using lipofectamine 2000 reagent.

Methods: Two methods of cell- and DNA-based heat treatment were used. Heating DNA solution was performed at 94 °C for 5, 10 and 15 min, and also 72 °C for 30, 60 and 120 min, individually. Moreover, heating the cells was done by incubation at 42 °C for 2 h in different times such as before, during and after DNA transfection.

Results: Our data showed that the conformation of plasmid DNAs was changed at different temperatures with increasing time. The heat-treated plasmid DNAs (94 °C for 10 min or 72 °C for 30 min) indicated higher transfection efficiency than untreated plasmid DNAs (p < 0.05). Furthermore, heat treatment of cells before and during the transfection was higher than untreated cells (p < 0.01). Our results demonstrated that DNA transfection efficiency in cancerous cells was less than non-cancerous cells (p < 0.01).

Conclusion: Generally, these findings showed that transfection mediated by thermal stimulation could enhance gene transfection in mammalian cell lines.

Keywords: Gene delivery; HIV-1; Heat shock protein; Heat treatment; Nef; Transfection.

Fig. 1

Schematic representation of transfection using heat-treated cells: The cells were incubated at 42 °C for 2 h before, during and after transfection. The transfection process was done for 4 h by adding 1 μg of plasmid DNA complexed with Lipofectamine 2000 into the cells. After the transfection, the medium was replaced and the cells were incubated for 48 h at 37 °C

Fig. 2

Confirmation of the recombinant plasmids by double-digestion in gel electrophoresis: The Nef (Lane 1) and Hsp27-Nef (Lane 2) genes were confirmed by restriction enzyme digestion as clear bands of ~ 648 bp and ~ 1368 bp on agarose gel, respectively

Fig. 3

Monitoring heat-denatured plasmid DNA on agarose gel: After denaturation of DNA at different temperatures and times, an extra band of DNA appeared (shown as arrow). Long-time heating process could break covalent bonds between double DNA strands

Fig. 4

Comparison of DNA transfection efficiency using thermal methods in HEK-293 T cell lines: A Heat-treated plasmid DNAs (94 °C for 10 min or 72 °C for 30 min) indicated higher transfection efficiency than untreated plasmid DNAs; B Heat treatment of the cells before transfection showed higher efficiency than untreated cells. The p values less than 0.05 were considered statistically significant; ns non-significant (p > 0.05)

Fig. 5

Comparison of DNA transfection efficiency using thermal methods in Vero (A), HeLa (B) and TC-1 (C) cell lines: Heat-treated plasmid DNAs (94 °C for 10 min or 72 °C for 30 min) indicated higher transfection efficiency than untreated plasmid DNAs. Moreover, heat treatment of the cells before transfection showed higher efficiency than untreated cells in Vero and TC-1 cell lines. However, DNA transfection efficiency in cancerous cells was less than non-cancerous cells. The p values less than 0.05 were considered statistically significant; ns non-significant (p > 0.05)