Psicor pgk puro vectors

ZBED1 and luciferase (negative control) shRNA constructs (shZBED1 and shLuc) were prepared as lentivirus using pSicoR PGK puro vectors (Addgene plasmid ID 12084). The shRNA sequence for ZBED1 (5′-GTGGCCATGTACATGCTCTAT) and luciferase (5′-CGCTGAGTACTTCGAAATGTC) were cloned into the cloning site of the vector, and the vectors were prepared as lentivirus by cotransfection with pMD2.G, pRSV-Rev, and pMDL g/p RRE (kindly provided by the Trono Lab through Addgene, Cambridge, UK) into HEK293T cells using PEI. After 20–24 h, the medium was changed to fresh DMEM medium with additives (10% FBS and 1% penicillin/streptomycin), and 48 h thereafter, the virus was harvested, filtered, and precipitated with PEG before resuspension in HAM’s F12 medium. For knockdown experiments, the virus was titrated to determine the amount needed for optimal ZBED1 knockdown and similar viral expression levels of shZBED1 and control shRNA by measuring the relative expression of ZBED1 and 5′-LTR 72 h after virus infection. For knockdown experiments, cells were seeded and the next day transduced with the respective shRNA construct in medium with the addition of 6 μg/mL of polybrene (Sigma-Aldrich). After 24 h incubation, the medium was changed to fresh medium, and after another 24 h, the transduced cells were set up for experiments.