Genetic manipulation of the BAC-cloned KSHV genome

To generate a series of KSHV helicase mutant KSHV genomes, mutagenesis of BAC16-RGB (RGB) was performed using a recombinant system as previously described. Briefly, a linear DNA fragment was amplified via PCR using the pEPKan-S plasmid as a template, which contained a kanamycin cassette, an I-SceI restriction enzyme site, and duplicated flanking sequences derived from KSHV genomic DNA (approximately 40-bp copy). The PCR product was treated with Dpn I to remove the plasmid template, and the purified PCR fragments were electroporated into the RGB-containing GS1783 strain, which had been induced at 42°C for 15 min. The recombinant clones were selected at 32°C on LB plates containing 12.5 μg of chloramphenicol and kanamycin (50 μg/ml). Positives clones were treated with 1% l-arabinose, induced at 42°C, and plated on LB plates containing 1% l-arabinose for secondary recombination. The resultant BAC was further confirmed through DNA sequencing.

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