293FE Cell Line

HEK293SF-3F6 (ATCC Accession No. CRL-12585) stably expressing the EBNA1 protein. The SFE cell line was developed by transfecting the 293SF-3F6 cell line with the pIRES-neo vector encoding the full-length EBNA1 protein and isolating and amplifying geneticin-resistant clones so obtained. The isolated clones were then tested for transient expression of the SEAP gene. A clone (clone 41) showing the highest expression levels was deposited under IDAC Accession No. 020502.

The cell line was cultured in HyQSFM4TransFx293 (HyQ) (Hyclone, Logan, UT, USA)  supplemented with 5% Cell Boost 5 (CB5, Hyclone).

Pre-cultures underwent passages every 2-3 days to keep the cells in their exponential growth phase. A few hours before transfection, the cell suspension was
centrifuged (300 g for 5 min) and re-suspended in fresh medium at 5 × 10^6 cell/mL. three different DNA concentrations
(0.4, 0.6, and 0.8 μg DNA/106 cells) were tested at 37°C.
The potential of repeated transfections at three different
times (0, 48, and 96 hours post-inoculation) and two
different temperatures (34 and 37°C) were also tested. In
the repeated transfection experiments, a new amount of
polyplexes was added at 48 and 96 hours, besides the
amount added in the beginning of the experiment