Constitution of the recombinant iSLK-RGB cell lines

The BAC-RGB plasmid was extracted using the NucleoBond Xtra Midi Kit. To generate KSHV helicase wild type or mutation recombinant virus–infected cells, 4 μg of the wild-type RGB or mutant RGB plasmids, respectively, was transfected into iSLK cells with 10 μl of the FuGENE HD Transfection Reagent. The medium of the iSLK cells was replaced with DMEM alone during transfection. At 1 hour after transfection, FBS was added to a final concentration of 10%. On the following day, the transfected iSLK cells were cultured in complete DMEM with a final concentration of hygromycin B (500 μg/ml) for selection. After 20 days of selection, hygromycin-resistant colonies were trypsinized, pooled, and subcultured. To induce viral lytic replication, the medium of BAC-carrying iSLK cells was replaced with fresh medium containing doxycycline (2 μg/ml) for 3 to 5 days.

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