Lenti x concentrator protocol

pLVX-TetOne-Puro (Clontech/Takara, 631,847) with puromycin selection was used as the backbone vector for an inducible gene expression system. Human PARG cDNA were cloned to pLVX-TetOne-Puro, followed by P2A cleavage site and mClover3 gene that encodes fluorescence protein with NLS signal for the visual control of the promoter activity.
Packaging Lenti-X Vectors into lentiviral particles and virus tittering: The lentiviral construct, pLVX-TetOne-hPARG-mClover3-NLS-Puro was packaged into lentiviral particles using Lenti-X Packaging Single Shots Protocol from Clontech/Takara. Lentiviral vector stocks were collected and concentrated using the Lenti-X Concentrator Protocol (Clontech/Takara). Lenti-X virus stocks were tittered using puromycin selection with HT1080 cells; the titer of virus corresponded to the number of colonies generated by the highest dilution multiplied by the dilution factor.
ccRCC cell lines were infected with lentivirus, selected with puromycin and single clones were generated using FACS sorting or with cloning rings. Clones with the highest fold change of hPARG induction vs. control detected with Western blotting were selected for propagation and further experiments.

For lentivirus production, 293 T cells were used and cultured in DMEM (Lonza) + 10% FBS. 293 T cells were transfected with FuGENE HD (Promega), MD2.G (envelope, Addgene), psPAX2 (packaging, Addgene), and FLAG-HA-U2AF1 and FLAG-HA-U2AF1^S34F vectors, and Opti-MEM (Thermo Fisher) overnight. The viral supernatant was collected after 48 h, centrifuged at 1500 × g for 45 min, and filtered through a 0.45 µm membrane. The resulting supernatant was concentrated overnight at 4 °C following the Lenti-X concentrator protocol according to the manufacturer’s instructions (Clontech). The following day, the lentivirus was centrifuged at 1500 × g for 5 min, resuspended in PBS at 1/100^th the original volume and stored at −80 °C.

The lentiviral construct, pLVX-TetOne-hPARG-IRES-mClover3-NLS-Puro was packaged into lentiviral particles using Lenti-X Packaging Single Shots Protocol from Clontech/Takara. Lentiviral vector stocks were collected and concentrated using the Lenti-X Concentrator Protocol (Clontech/Takara). Lenti-X virus stocks were tittered using puromycin selection with HT1080 cells; the titer of virus corresponded to the number of colonies generated by the highest dilution multiplied by the dilution factor.