To produce high-titer lentivirus, we used standard protocols. Briefly, about 1.2 × 107 HEK293T cells were plated in 15-cm cell culture dishes in 25 mL Dulbecco’s MEM supplemented with 10% FBS. The next day, cells were transfected with Lipofectamine3000 (Invitrogen) DNA mixture (10 µg of pLKO.1 shRNA plasmid/10 µg of pLOC-DCN, 7.5 µg of psPAX2 packaging plasmid and 2.5 µg of pMD2.G envelope plasmid) and were incubated overnight. The culture medium was then removed and replaced with fresh medium. The supernatant containing the virus was then collected, filtered through a 0.45 μm HV Durapore membrane (EMD Millipore, Burlington, MA, USA) to remove cells and large debris, and concentrated by ultracentrifugation.
For transduction, target cells were about 70% confluent. Two hours before transduction, the medium was changed, and then transductions were carried out for 24 h in the presence of 8 μg/mL polybrene (Sigma-Aldrich). Cells expressing fluorescent protein (GFP/RFP) were sorted by fluorescence-activated cell sorting and expanded before in vitro and in vivo experiments.
For transduction, target cells were about 70% confluent. Two hours before transduction, the medium was changed, and then transductions were carried out for 24 h in the presence of 8 μg/mL polybrene (Sigma-Aldrich). Cells expressing fluorescent protein (GFP/RFP) were sorted by fluorescence-activated cell sorting and expanded before in vitro and in vivo experiments.