title: Efficacy and site-specificity of adenoviral vector integration mediated by the phage φC31 integrase.
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 φC31 integrase (φC31-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 φC31-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 φC31-Int. In conclusion, integration of HD mediated by φC31-Int is an attractive alternative to engineer cells, because it permits site-specific integration of large DNA fragments with low genotoxicity.
authors.affiliation: Biotechnology Research Institute, National Research Council Canada, Montréal, Canada, H4P 2R2.
authors.forename: Marc-André
authors.initials: MA
authors.lastname: Robert
authors.forename: Yue
authors.initials: Y
authors.lastname: Zeng
authors.forename: Benoît
authors.initials: B
authors.lastname: Raymond
authors.forename: Laurie
authors.initials: L
authors.lastname: Desfossé
authors.forename: Emilie
authors.initials: E
authors.lastname: Mairey
authors.forename: Jacques P
authors.initials: JP
authors.lastname: Tremblay
authors.forename: Bernard
authors.initials: B
authors.lastname: Massie
authors.forename: Rénald
authors.initials: R
authors.lastname: Gilbert
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001435:Bacteriophages / Q000201:enzymology*; D001483:Base Sequence; D002460:Cell Line; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000235:genetics*; D051379:Mice; D014161:Transduction, Genetic
chemical_list: D006657:Histones; D019426:Integrases