Generation of stable 293T cells carrying BAC36 and BACΔ6 genomes.

(A) Transfection of 293T cells with BAC36 and BACΔ6 DNAs. Cells were transfected with BAC36 (1 and 3) and BACΔ6 (2 and 4) with lipofectamine 2000. GFP expression levels were monitored by fluorescent microscopy 2 days post-transfection (1 and 2). Then, the transfected cells were split and selected with hygromycin. Hygromycin-resistant clones of 293T-BAC36 and 293T-BACΔ6 cells were established (3 and 4). (B) RT-PCR confirmation of the absence of ORF6 in 293T-BACΔ6 cells. Total RNA from uninduced or TPA/NaB induced 293T-BAC36 cells and 293T-BACΔ6 cells were used to prepare cDNAs. The RT-PCR generated products were analyzed on a 1.5% agarose gel. Shown are the KSHV ORFs 6, 7, 73, 50, 59 and 65, respectively. β-actin was used as the normalization control for input RNA. Absence of contaminating DNA in the samples was tested by reverse transcriptase-negative control reactions (RT control). (C) Western blot confirmation of the absence of ORF6 in 293T-BACΔ6 cells. The 293T-BAC36 cells and 293T-BACΔ6 cells were induced with TPA/NaB for 2 days. Whole cell lysates were immunoblotted with antibodies against ORF6, LANA, RTA, ORF45 and ORF59. The same blots were probed with anti-β-actin antibody to ensure equal loading of all samples. (D) Real-time PCR analysis of the mRNA levels of ORF4 and ORF7 from 293T-BACΔ6 cells induced with TPA/NaB. cDNAs were prepared as described previously and analyzed by real-time PCR using primers specific for ORF4 and ORF7 transcripts. β-actin was used as an internal standard. The data are shown as the fold increase compared to the untreated 293T-BAC36 cells.