Figure 4

In vivo cleavage of human telomere by chimeric endonucleases. We expressed chimeric endonucleases (G-T, EN-T, FN-T) mediated by adenovirus (MOI: 100) and collected genomic DNA 3 days after infection (see Methods). G-T, GFP-TRF1; -, no virus infection. Using telomeric repeats as a probe, we performed Southern hybridization for terminal restriction fragment (TRF) assay in order to analyse telomere length. We used L1 probe (the sequence is shown in Additional file 6) (a, c and d), and alpha satellite probe (b) as an internal control. (e) Telomere digestion by chimeric endonucleases in U2OS cells. We added each virus construct at MOI 100, 10, 1 (left to right lanes) to the cells, and performed a TRF assay with the same procedure as in (a). G, GFP; other abbreviations (see Figure 1a or text). (f) Protein expression in U2OS cells after adenoviral infections. Expressed proteins used in (e) were confirmed by western blotting using anti-HA antibody. The amounts of expressed proteins are directly proportional to viral titers.