Figure 6

Detection of intact provirus DNA in the DSB site. (A) I-PpoI-qPCR screening of cell clones containing provirus DNA in I-PpoI site. HT1080 cells were infected with Ad-I-PpoI at an MOI of 30 in the medium with 0.1% FBS. After 24 h, cells were further infected with lentiviruses (VSVG-pseudotyped Lenti6-EGFP-D64V) also under serum-starved conditions. Two h later, medium was changed with fresh one with 0.1% FBS. On the next day, medium was replaced with a complete medium with 10% FBS. Blasticidin-resistant colonies were isolated and I-PpoI site targeting provirus was detected by I-PpoI-qPCR. The threshold of detecting provirus integrated as direct or inverted repeat orientation was −1 log(10) copies/cell (indicated in red horizontal lines). (B) EGFP expression analysis. Cells containing the proviral DNA in I-PpoI site in (A) were further analyzed for the expression of EGFP by flow cytometer. (C) The estimation of proviral DNA copy number. Copy numbers of provirus DNA in EGFP-positive clones, shown in (B), were analyzed by Southern blot by using a part of DNA fragment of the lentiviral vector as a probe. Genomic DNA extracted from each clone was digested with BamHI or EcoRI prior to electrophoresis. Restriction maps are shown (right panel). B, BamHI; E, EcoRI; P, I-PpoI. Of note, clone #2413 possessed a single copy of provirus DNA. (D) Sequence analysis of lentiviral vector integrated in the I-PpoI site. EGFP-positive clones shown in (B) were subjected to sequence analysis. I-PpoI-PCR amplicons were directly used as a template for sequence analysis. (E) FISH analysis of the #2413 clone. (F) Nucleotide sequence of intact proviral DNA present in the DSB site. The proviral DNA of #2413 clone was sequenced and whole nucleotide sequence data was shown. In #2413 clone, no structural alternations of provirus DNA were detected.