Construction of the dual regulated oncolytic adenovirus AD55-Apoptin and its selective replication in tumor cells. A. Schematic structure of recombinant adenoviruses. Compared with E1B 55KD-deficient adenovirus ZD55, the dual-regulated adenovirus AD55-Apoptin had been further modified with both the E1A promoter replaced by eAFP and the E1b55KD deletion. Then, the Apoptin expression cassette controlled by human cytomegalovirus (HCMV) promoter was obversely inserted to form AD55-Apoptin. ITR is the inverted terminal repeat sequence. B. Detection of E1A levels of recombinant oncolytic adenoviruses. L-02, Huh-7 and PLC was infected with ONYX-015, AD55 and AD55-Apoptin at the MOI of 5, after 48 hours, Western Blotting was conducted to detect E1A protein expression, β-actin was used as a protein loading control. C. 3.5 × 105 cells were plated into six-well plates. After 24 h, the cells were infected with 10 MOIs of AD55-Apoptin or AD55 or ONYX-015 or ZD55-EGFP, respectively. After an additional 48 h, medium and cells were scraped into 1.5 ml Eppendorf tube and subjected to three-thaw cycles. The collected supernatant was tested for virus production by standard TCID50 assay on 293 cells. Progeny viruses from 1 MOI of virus were calculated. The results were the average of two independent experiments. D. Huh-7 and QSG-7701 cells were infected with AD55 or AD55-Apoptin at the MOI of 10 for 24, 48 and 96 hours, respectively. The total RNA was collected at the indicated time and reverse transcripted into cDNA, then a real time quantitative PCR was done with the Apoptin or GAPDH primers.