FIGURE 5.

KSHV BAC-16 recombineering. A, shown is a schematic depiction of the recombineering procedures used for construction of BAC-16 LANA mutants. B, establishment of 293T BAC stable cell line shows >90% GFP-positive cells after hygromycin selection. FITC channel (GFP, left) and phase (right) images are shown. C, Southern analysis used recombinant KSHV bacmid DNA that was digested with BamHI and probed as indicated. Analysis of BAC-16 LANA R20K allele is shown. D, shown is the nucleotide sequence near the LANA initiator methionine of the parental BAC-16 (upper sequence) compared with a bacmid that had undergone successful recombineering (lower sequence). The LANA ATG is underlined. The single residual 34-bp FRT site plus 12 bp derived from the targeting vector are shown at the insertion site between nucleotides −1 and +1 relative to the LANA ATG.