Padenovator cmv5 ires gfp shuttle vector

Expression vectors encoding the hTERT-specific ribozyme and HSVtk gene at the 3′ exon with or without post-transcriptional regulators including enhancer elements (SA/SA and/or WPRE) and three copies of 122T under the control of the CMV or PEPCK promoter were cloned into the pAdenoVator-CMV5-IRES-GFP shuttle vector (Qbiogene, Irvine, CA, USA) to generate recombinant adenoviral vectors. Recombinant adenovirus vectors encoding the ribozymes were created by a homologous recombination technique in bacteria (BJ5183) as follows. In brief, the shuttle plasmid was linearized with PmeI and subsequently co-transformed into BJ5183 cells with an E1/E3 deleted adenoviral type5 backbone genome (pAdenoVator DE1/E3; Qbiogene). The recombinant vectors generated through homologous recombination in the bacteria were isolated and linearized with PacI. The linearized vectors were then transfected into HEK293 cells, and the recombinant adenoviruses produced were isolated by three rounds of plaque purification. Recombinant viruses were amplified, purified, qualified, and concentrated via ultracentrifugation (Beckman-Coulter, Brea, CA, USA). Titers of the recombinant adenovirus were determined by TCID50 analysis.