Transient transfection of CHO cells using linear polyethylenimine is a simple and effective means of producing rainbow trout recombinant IFN-γ protein

Abstract

A practical method was developed for the transient transfection of Chinese hamster ovary (CHO) cells with 25 kDa linear polyethylenimine (PEI) then optimal culture conditions determined for the production of rainbow trout (Oncorhynchus mykiss) IFN-γ recombinant protein. We found that culture temperature had a significant impact upon recombinant protein yield, with best results being obtained at 32 °C. However the amount of serum added to the culture medium had no effect upon recombinant IFN-γ (rIFN-γ) production. In this study maximal rIFN-γ yields and minimal PEI toxicity were achieved using a DNA/PEI ratio of 1:8, where the amount of PEI did not exceed 10 µg per 5 ml of RPMI1640 culture medium, with cells subsequently cultured at 32 °C for 7 days. Thus, linear PEI is a technically simple and cost-efficient method for the transient transfection of CHO cells and is compatible with serum-free operations.

Keywords: Chinese hamster ovary cells; Linear polyethylenimine; Rainbow trout IFN-γ; Recombinant protein; Transient transfection.

Fig. 1

Analysis of toxicity of PEI. The dose of PEI tested ranged from 0 to 40 µg, with the ratio of DNA/PEI fixed at 1:8. All cells of each sample, including those in the supernatant and wash buffer, were collected and counted. The percentage of dead cells was assessed using a Neubauer counting chamber with trypan blue exclusion. Data represent means + SEM of four independent counts from four representative fields for each sample

Fig. 2

Assessment of transfection efficiency of CHO cells by PEI. a The images of CHO cells transfected with the pmaxGFP vector. Observations were made by fluorescence microscopy of CHO cells transfected with pmaxGFP at a 1:8 ratio of DNA/PEI at 48 h post-transfection (magnification at ×20). The left panel (I) is a brightfield image of the cells. The middle panel (II) shows GFP positive CHO cells observed under fluorescence. The right panel (III) is an overlay of images of I and II to show the percentage GFP positive CHO cells. b Flow cytometry analysis of CHO cells transfected with pmaxGFP at 48 h post-transfection. Untransfected cells were used as control. The efficiency analysis was performed three times with both transfected and untransfected cells in three wells, respectively

Fig. 3

Estimation of rIFN-γ protein yields at different DNA/PEI ratios. a The standard curve used to calculate the amount of recombinant protein present in a supernatant sample. RTG-3F7 cells were incubated with a serial dilution of rIFN-γ produced by E. coli. Then the luminescence was measured to build the standard curve. b The yield of recombinant protein from CHO cells transfected at different ratios of DNA/PEI. RTG-3F7 cells were incubated with the rIFN-γ containing supernatants produced by CHO cells. The CHO cells were transfected with different ratios of DNA/PEI with the PEI fixed at 10 µg of 5 ml of culture medium while empty pEF-LacZ expression vector was used as a control (CK), then the cells cultured for 3 days at 37 °C before supernatant harvest. Luminescence was detected in a Steady Glo Luciferase assay. ***P < 0.001

Fig. 4

Estimation of rIFN-γ protein yields at different temperatures. a The comparison of the yields of recombinant IFN-γ protein from transfected CHO cells cultured at 32 or 37 °C with a fixed FCS concentration of 0.5 %. b Comparison of rIFN-γ protein yields from transfected CHO cells cultured at 32 or 37 °C with a fixed FCS concentration of 10 %. The supernatants were collected at days 2, 4, 7 and 14. The yields were calculated according to the formula from the standard curve. The results are presented as averages + standard error for three treatments. Asterisks indicate significant differences to day 2 (*P < 0.05). Daggers indicate significant differences between 32 and 37 °C on the same day (^† P < 0.05; ^†† P < 0.01)

Fig. 5

Estimation of rIFN-γ protein yields in different concentrations of FCS. a Comparison of rIFN-γ yields of transfected CHO cells cultured under 0.5 or 10 % FCS at 32 °C. b The comparison of the yields of IFN-γ of transfected CHO cells cultured under 0.5 or 10 % FCS at 37 °C. The supernatants were collected at day 2, 4, 7 and 14. The yields were calculated according to the formula from the standard curve. The results are presented as averages + standard error for three treatments