Advanced Adenovirus Generation
Applied Viromics utilizes E1/E3-deleted recombinant Adenovirus Serotype 5 (Ad5) vectors, which are replication-deficient and offer highly efficient gene transfer in both dividing and non-dividing mammalian cells.
Helper-Virus Free System: Our optimized packaging system completely eliminates helper virus co-infection, removing any risk of helper virus contamination in the final formulation.
Four-Step Adenovirus Packaging Protocol
Adenovirus vector production requires initial plasmid transfection, followed by sequential viral amplification to generate sufficient titers for density gradient purification.
Subcloning & Vector Design
Your Gene of Interest (GOI) is subcloned into our pAd-shuttle vector. The plasmid is linearized and prepared for transfection.
Primary Seed Generation (P0)
HEK293 cells (supplying the essential E1 genes in trans) are co-transfected with the pAd-shuttle plasmid and E1/E3 helper plasmids. Primary P0 Adenovirus is harvested after cytopathic effect (CPE) develops in approximately 2 weeks.
Viral Amplification (P1 & P2)
To scale up the viral volume, HEK293 cells are infected with the P0 seed to generate the P1 stock. The P1 stock is amplified to yield the high-titer P2 generation, which serves as the production harvest.
CsCl Purification & Dialysis
The P2 harvest is lysed and purified using double Cesium Chloride (CsCl) density gradient ultracentrifugation, yielding highly pure, high-concentration adenoviral preparations. A desalting chromatography step exchanges the virus into optimal formulation buffer.
Insert Limit
The packaging limit of our E1/E3-deleted pAd-shuttle vector is 5.3 kb. Genes exceeding this size cannot be packed effectively into the virion capsid.
Storage Temp
Adenoviruses lose half their activity when stored at 4°C for 7 days. Aliquot upon receipt and store strictly at -80°C for long-term stability.
Safety Profile
According to NIH guidelines, E1-deleted Adenoviruses are Risk Group 1 agents. They can be safely handled in a **Biosafety Level 1 (BSL-1)** environment.