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Construction, propagation, and titer estimation of recombinant adenoviruses carrying proapoptotic genes
N Shinoura1, M Ohashi, Y Yoshida
1Department of Molecular Biotherapy Research, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo.
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.
Insights
Generating recombinant adenovirus (Adv) with apoptotic genes is challenging. Using CrmA-transduced 293 cells as hosts enables efficient production of Adv carrying proapoptotic genes like Fas and Fas ligand.
Area of Science:
- Molecular Biology
- Virology
- Gene Therapy
Background:
- Recombinant adenovirus (Adv) generation is hindered by host cell apoptosis.
- Apoptotic damage limits the production of Adv carrying apoptosis-inducing genes.
Purpose of the Study:
- To develop an efficient method for generating recombinant Adv with proapoptotic genes.
- To overcome the limitations of host cell apoptosis during Adv construction.
Main Methods:
- Transduction of 293 cells with the caspase-inhibiting CrmA gene to create 293-CrmA host cells.
- Utilizing 293-CrmA cells for the construction of Adv encoding Fas and Fas ligand.
- Employing Adv DNA copy number determination to quantify infective virus.
Main Results:
- 293-CrmA cells demonstrated high efficiency in constructing recombinant Adv with Fas and Fas ligand.
- Ample quantities of recombinant viruses were produced using the 293-CrmA cell line.
- Adv DNA copy number assay proved necessary for accurate infective virus quantification.
Conclusions:
- The 293-CrmA cell line is a robust platform for producing recombinant Adv carrying proapoptotic genes.
- The described techniques facilitate ample production and accurate quantification of recombinant Adv.
- This approach is broadly applicable for Adv-based gene therapy vector development.