Lentivirus-Mediated Gene Manipulation in Thyroid Cancer Cells

Using human PC gene primer sequences, PCshRNA-GFP-puro-lentivirus, scramble-GFP-puro-lentivirus, PC-OE-GFP-puro lentivirus, and vector-GFP-puro were purchased from Hanheng Biotechnology (Hanheng Biotechnology Co., Ltd., Shanghai, China). We previously injected various concentrations of GFP-labeled lentivirus into the cells and measured the effectiveness of the infection, and then applied the obtained optimal multiplicity of infection in subsequent lentivirus infection experiments [44 (link)]. TPC-1 and KTC-1 cells were infected with lentiviruses by plating in six-well plates to reach 60–80% confluence, followed by the addition of 1 mL RPMI-1640 complete culture medium containing 2 mg/mL polybrene (Hanheng Biotechnology Co., Ltd.). Lentiviral supernatant (50 μL) was then collected and added to the dish, followed by 1 mL of culture medium 4 h later. After 48 h, the culture medium was replaced with RPMI-1640 complete medium containing 1 mg/mL puromycin (Sigma-Aldrich, St. Louis, MO, USA) and cultured for 1 week for screening.