Recombinant non replicative lentiviral plasmid

MCF7 and T47D cells were infected with a recombinant non-replicative lentiviral plasmid (Sigma, St. Louis, MO, USA) containing human shGPR81 (transfected with 2 different shRNA constructs for GPR81) or with a control plasmid (pLKO.1-puro) obtained from Sigma. Each construct was co-transfected with the packaging constructs VSVG (viral glycoprotein expression vector) and delta 8.9 (packaging vector) packaging constructs. Lentivirus was produced in 293T cells using the LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA, USA). The medium was replaced at 16 h after transfection, and the supernatant was harvested after an additional 48 h. The lentiviral particles were used to transduce the target cells for 24 h. The cells were infected with lentivirus (500 μl of supernatant/ml medium) mixed with polybrene (4 μg/ml). Puromycin-resistant clones (3 μg/ml) were then isolated using the limiting dilution method. GPR81 knockdown was verified by RT-PCR and Immunocytochemistry.