Adenovirus Packaging And Amplification

Our viral vector team has outlined the basic major steps in adenoviral amplification. Note, the below protocol is not exhaustive, but used only to provide a general overview on the adenovirus amplification process.

1. Initial Transfection
   • A recombinant adenoviral plasmid containing the desired transgene is transfected into a permissive cell line such as HEK293T cells. Learn more about adenovirus packaging cell line.
   • HEK293T cells supply essential viral replication proteins missing from the recombinant adenovirus genome.
2. Production of Virus
   • The transfected cells produce the first generation of recombinant adenoviruses, which are released upon cell lysis.
   • This crude viral lysate contains a low concentration of adenoviral particles.
3. Infection for Amplification
   1. Crude lysate is used to infect a larger batch of HEK293 cells.
   2. As the virus infects the cells, it hijacks the cellular machinery to replicate and package more viral genomes, producing large quantities of virus.
4. Adenovirus Harvesting
   1. When most cells show cytopathic effects (CPE), indicating they are infected and have produced virus, the cells and culture medium are collected.
   2. Cells are lysed to release the viral particles, which can then be purified.
5. Viral Vector Purification
   1. The virus is typically purified using  ultracentrifugation to remove cellular debris and concentrate the viral particles.
6. Quantification
   1. The viral titer is determined by RT-qPCR assay to assess the concentration of infectious viral particles.