Construction, Propagation, and Titer Estimation of Recombinant Adenoviruses Carrying Proapoptotic Genes

Abstract

Generation of a recombinant adenovirus (Adv) that induces the constitutive expression of an apoptotic gene has been extremely difficult owing to severe apoptotic damage to the host cell. In this study, 293 cells were transduced with the caspase-inhibiting CrmA gene (293-CrmA cells), and used as host cells to generate Adv carrying apoptosis-inducing genes (proapoptotic genes). The 293-CrmA cells proved to be highly efficient for the construction of recombinant Adv carrying genes encoding Fas and Fas ligand. Moreover, the 293-CrmA line produced an ample quantity of these recombinant viruses. Because the conventional 293 plaque formation assay did not reflect the actual number of cells infected with the Adv carrying the proapoptotic gene, a determination of the Adv DNA copy number introduced into target cells was necessary to evaluate the quantity of infective virus. The techniques described here should be widely applicable for the construction of a recombinant Adv, in ample quantity, and for the estimation of the quantity of recombinant Adv produced. Recombinant adenoviruses (Advs) carrying proapoptotic genes, such as those encoding Fas and Fas ligand, will be useful tools for basic biological studies of immunity and oncogenesis, as well as in applications for cancer gene therapy. However, the damage to the host cells incurred by proapoptotic genes causes difficulties in construction and propagation of these Advs. In this study, we have developed an efficient method for the construction and ample propagation of Advs by using sublines of 293 cells transfected with the caspase-inhibiting gene. Because the titration of Advs carrying proapoptotic genes by plaque formation assay greatly underestimated the quantity of Advs, the introduced Adv DNA copy number should be an important parameter in the estimation of viral titer.