Establishing Stable Cell Lines with shRNA

The LLC-PK1 cells were seeded (2 × 10⁴/well) into 6-well plates and incubated for 24 h at 37 °C in a 5% CO₂ atmosphere. When reaching 50–70% confluence, the cells were washed three times with sterilized 0.01 M pH7.4 phosphate buffered saline (PBS). Afterwards, the cells were transfected with 2.5 μg/well of shRNA-expressing plasmids using a Lipofectamine^TM 3000 (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. After 24 h of incubation, the growth media were substituted with maintenance media containing 2% FBS and 1000 μg/mL of Neomycin (G418). The survival cell clones were maintained in G418-containing media for 15 d with frequent media replacements until cell death could no longer be observed. Then, these monoclonal cells transfected with shRNA-expressing plasmids were screened by limiting dilution analysis (LDA), as previously described [29 (link)], and cultured in DMEM growth media containing G418 (500 μg/mL) in 6-well plates at 37 °C in a 5% CO₂ atmosphere. After reaching a confluence, these plasmid-transduced cells were inoculated with PDCoV at a multiplicity of infection (MOI) of 0.1. Non-transfected cells were set as a control. Cell transfection efficiency and cytopathic effect (CPE) images were taken under an inverted fluorescence/phase-contrast microscope (Nikon, Tokyo, Japan).