Single plasmid-based, upgradable and backward compatible adenoviral vector systems.

Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, we constructed single plasmid-based adenoviral vector systems where a unique PmeI site was located at the position for insertion of exogenous gene. PCR amplified transgene could be cloned into PmeI-linearized start plasmids by one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was designated restriction-assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded start plasmid pKAd5f11pABR-EPG. Modified fiber gene, amplified by overlap extension PCR, could be used to substitute original fiber in pKAd5f11pABR-EPG to generate adenoviral plasmid with new fiber by restriction-assembly. On the other hand, pKAd5f11pABR-EPG was also a start adenoviral plasmid for expressing other transgene. In conclusion, we introduced easy-to-use and upgradable adenoviral vector systems here, which offer extensive versatility and can serve as basic platform and functional component library for the synthetic biology of adenoviral vectors.