Construction of a recombinant adenovirus carrying IL- 12 -IRES-CK gene and its expression in hepatocyte

AIM: To construct a bicistronic recombinant adenovirus carrying creatine kinase (CK) and human interleukin 12 (hIL-12) gene, and then detect the expression of both genes in vitro and metabolic product of CK in vivo. METHODS: Two PCR products, CK and hIL- 12 genes linked by IRES, were inserted into adenoviral vector. Adenovirus particles carrying CK-IRES-IL- 12 gene was generated through homologous recombination, packaging and propagation. Rabbit hepatocytes were isolated and transfected with adenovirus particles. CK and IL-12 gene expression was confirmed by Western blotting and ELISA, respectively.RESULTS: The expression of CK and human IL-12 in hepatocyte were confirmed in vitro. Metabolic product of CK, phosphocreatine (PCr), could be detected in liver by non-invasive phosphorus-31 magnetic resonance spectroscopy (³¹P MRS) in vivo.CONCLUSION: In vivo detection of ectopic expression of CK gene in liver can be achieved by non-invasive ³¹P MRS through adenovirus transduction. The bicistronic recombinant adenovirus Ad5-hIL-12-IRES-CKb carrying CK and hIL- 12 provides a useful tool for the further study of using a reporter gene expression (CK) dynamically, non-invasively monitor a therapeutic gene expression (IL-12) in liver.