2.4. Transduction and Puromycin Selection of BHK-21 Cells

BHK-21 cells were transduced by recombinant lentiviruses according to the methods described previously [23]. Briefly, BHK-21 cells were plated in 24-well plates (Corning, Corning, NY, USA) and grew overnight until the cultures reached about 60% confluence. The cells were rinsed, and each well received 450 µL of cell culture medium, 50 µL of recombinant lentiviruses stocks, and 3 µg of polybrene (Inovogen Tech. Co., Ltd., Chongqing, China). The plate was placed in a 37 °C/5% CO₂ incubator for 24 h. Then, the media was replaced with DMEM containing 10% FBS, and cells were cultured for an additional 24 h. Forty-eight hours after transduction, the transduced BHK-21 cells were trypsinized and seeded in 6-well plates (Corning, Corning, NY, USA) at a density of 100~200 cells/well, cultured with fresh DMEM containing 10% FBS and 5 µg/mL puromycin (Inovogen Tech. Co., Ltd., Chongqing, China) for selection. The selective medium was exchanged every 3~4 days for up to three weeks until nearly all of the EGFP-negative cells were killed. The surviving cell colonies were screened and sub-cloned at least three times by the limiting dilution method. Finally, two positive BHK-21 cell lines that expressed the N protein of AKAV were established and were designated as C8H2 and F7E5, respectively.