Figure 2. Absence of viral integration in Adeno-iPS cell.

(A) Schematic drawing of the adenoviral vector indicating the position of the cDNA and the sizes of the respective DNA fragments after BamHI digestion. The bracketed BamHI site is only present in the Oct4 cDNA. A pBluescript (pBS) sequence present in both the adenoviral vector and the Oct4^IND transgene is highlighted. (B) PCR analyses for adenoviral integration in genomic DNA from the indicated Adeno-iPS clones as well as from V6.5 ES cells that served as a negative control (−). Arrowhead indicates the position of the positive control band amplified from vector DNA (+). (C) Southern blot analysis of BamHI-digested genomic DNA using DNA fragments encompassing the entire adenoviral vector backbone isolated from pAd-Oct4 as probes. Plasmid DNA of pAd-Klf4 and pAd-Oct4 diluted to the equivalent of 0.2, 1 or 2.5 integrations per genome and genomic DNA of HEK cells (which contain adenoviral sequences in their genome) were used as positive controls. An asterisk (*) indicates bands resulting from hybridization of the pBS sequence in the adenoviral probe to transgenic sequences in the Oct4^IND allele. Note that these bands are absent in HEP iPS, V6.5 ES and HEK cells. A double asterisk (**) indicates bands resulting from hybridization of parts of the probe to sequences present in the endogenous Oct4 locus or in Oct4 pseudogenes. These bands are present in all lanes containing genomic DNA including the wild type control and therefore serve as loading controls. Solid arrowheads indicate the position of BamHI fragments of the adenoviral vector and open arrowheads highlight adenoviral sequences detected in HEK cells.