To construct the IRF7 expression vector, the fragments encompassing the IRF7 sequence were synthetized chemically, and then cloned into the BamHI and AgeI sites in lentiviral vector (GV358) (Genechem, Shanghai). Plasmid DNA was transformed in MAX Efficiency® DH5a™ Competent Cells (Invitrogen, Mount Waverley, Victoria, Australia) as described by the manufacturer. Positive colonies were further cultured for plasmid amplification and purification using the Wizard® Plus Midiprep DNA Purification System (Promega Corporation) as per manufacturer's instructions. Purified plasmid DNA was verified for the presence of IRF7 insert by restriction digest with BamHI/AgeI. All constructs were further confirmed by DNA sequencing analysis.
293T cells were cultured at 10cm culture dishes for 24h, before the cells were co-transfected with GV358-IRF7 DNA (20μg), pHelper 1.0 (15μg), and pHelper 2.0 (15μg) using Lipofectamine 2000 in 1ml volume for 6hr (Invitrogen). After co-transfection for 6hr, the transfected supernatants were removed, and the dished were washed once by using 10ml 1×PBS, and added new cell medium. The culture supernatants were collected after the 293T cells cultured for 48hr and used as virus stock after concentration. Viral titer was determined by HIV-1 P24 Antigen ELISA (ZeptoMetrix Corporation) after infection.
293T cells were cultured at 10cm culture dishes for 24h