Adenovirus has the ability to accommodate up to ~7.5 kb of cargo sequence making it an ideal choice for researchers to express larger open reading frames (ORFs) with high infection efficiency. There are over 51 different serotypes in human, the most used type for research is human adenovirus type 5. The recombinant adenovirus can infect dividing and non-dividing cells, support transient, high-level protein expression, and are also easily amplified and purified to high concentrations. The Adenovirus genome is double-stranded DNA of approximately 36kb. It contains four early-transcribed regions E1, E2, E3 and E4. The E1 gene products, including E1A and E1B, are involved in replication of the virus. The E2 proteins provide the machinery for viral DNA replication and transcription of late genes. Most of the E3 proteins are involved in modulating the immune response of infected cells. The E4 gene products are involved in the metabolism of virus messenger RNA and provide functions that promote virus DNA replication and shut-off of host protein synthesis. To make the adenovirus an effective and safe tool for gene delivery, two genes involved in viral replication (E1) and modulating host immune response (E3) are deleted (E1/E3 genes deleted) in human adenovirus type 5.
Syno® Adeno virus Production Procedure
Recombinant adenovirus packaging requires two vectors: the shuttle vector containing the gene of interest (GOI) and the recombinant adenovirus vector (pAD). The first step of adenovirus production involves transferring the GOI from the shuttle vector to pAD through recombination of the left and right homology arms. The recombinant adenovirus vector containing the GOI is then linearized and the packaging cell line (HEK293T cells expressing E1) is transfected with the linearized construct to generate the primary stock of recombinant adenovirus. This primary stock is then harvested and amplified through multiple rounds of infection. The viral particles are then purified using gradient ultracentrifugation to remove impurities and ensure safe use for in vitro or in vivo research.