| Abstract | Adenoviral vectors deleted of all their viral genes (helper-dependent [HD]) are efficient gene-transfer vehicles. Because transgene expression is rapidly lost in actively dividing cells, we investigated the feasibility of using phage C31 integrase (C31-Int) to integrate an HD carrying an attB site and the puromycin resistance gene into human cells (HeLa) and murine myoblasts (C2C12) by co-infection with a second HD-expressing C31-Int. Because the HD genome is linear, we also investigated whether its circularization, through expression of Cre using a third HD, affects integration. Efficacy and specificity were determined by scoring the number of puromycin-resistant colonies and by sequencing integration sites. Unexpectedly, circularization of HD was unnecessary and it even reduced the integration efficacy. The maximum integration efficacy achieved was 0.5% in HeLa cells and 0.1% in C2C12 myoblasts. Up to 76% of the integration events occurred at pseudo attP sites and previously characterized hotspots were found. A small (two- to three-fold) increase in the number of γ-H2AX positive foci, accompanied by no noticeable change in γ-H2AX expression, indicated the low genotoxicity of C31-Int. In conclusion, integration of HD mediated by C31-Int is an attractive alternative to engineer cells, because it permits site-specific integration of large DNA fragments with low genotoxicity. |
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