Establishment of cell lines stably expressing Gag-TRAP-GFP

U373MG stable cell lines were established using the Murine Stem Cell Virus (MSCV)-based retroviral vector system. Recombinant retroviral vectors were generated by transfection of 293T cells with the pMSCV-neo vector encoding Gag_1-200-RevNoLS-GFP (Gag-TRAP-GFP), Gag_1-200-GFP, RevNoLS-GFP or GFP, and the packaging plasmids expressing MLV Gag-Pol and the Vesicular Stomatitis Virus envelope G glycoprotein (VSV-G) using the calcium phosphate precipitation method. Cell-free supernatants were collected 48 h post-transfection and used to transduce U373MG cells. GFP expression was analyzed 48 h post-transduction by flow cytometry. After cell sorting, GFP-positive cells were propagated in culture medium supplemented with G418 (500 µg/mL).