Traditionally, recombinant adenovirus was made through homologous recombination between a shuttle plasmid and a full-length Ad backbone. This method could take 2 months or longer and often required extensive plaque purification to eliminate wild-type contaminants. The rapid system uses a simplified protocol that produces high titer adenovirus within 2-3 weeks and uses a novel Ad backbone that is devoid of the left-hand ITR, packaging signal and E1 sequences.
Removal of these unnecessary adenoviral sequences reduces or eliminates the production of wild-type viruses, eliminating the need for plaque purification. Generating adenovirus with the RAPAd system involves linearizing the shuttle and backbone vectors, cotransfecting into 293 cells, and harvesting the virus within 15 days. Once the virus stock has been harvested it can be amplified using 293 cells.
The first step in generating a gene-specific recombinant adenovirus is cloning the gene of interest into the shuttle vector. The shuttle and backbone vectors are then linearized and co-transfected into 293 cells using the desired transfection reagent. The viral crude lysate is collected 7-10 days after transfection, once greater than 50% of the cells have lifted from the plate. The virus is harvested by performing a series of freeze/thaw cycles and can then be stored, tittered, amplified, or purified.
A unique feature of adenovirus is the ability of recombinant adenoviral stocks to be amplified using 293 cells, allowing for propagation of the viral stock without having to repeat the transfection step. Amplification can also be performed to increase the titer. To amplify adenovirus, the crude or purified lysate is added directly to 293 cells and incubated until cytopathic effects (CPE) are observed. The cells are then harvested, the virus released with freeze/thaw cycles, and the supernatant is collected as a high titer viral stock.
Accurately quantifying the amount of virus present is important for ensuring that enough virus is delivered to the target cells and for maintaining consistency between experiments. The viral titer should be measured once the virus is packaged and amplified, and again after any purification or concentration procedures. Adenovirus titer can be measured by plaque assay, which can take up to 14 days, or by using a titer kit that can be completed within 2-3 days.
When using adenovirus for in vivo studies or if the crude lysates are toxic to target cells, purification is recommended to remove the cellular debris that can interfere with infection. CsCl ultracentrifugation can be used for purification, or a purification kit can be used which will recover up to 90% of the virus. The purified lysate should be re-tittered.
Once the virus is packaged and tittered, it is ready to be applied to target cells. The appropriate multiplicity of infection (MOI) should be determined for each target cell line using a GFP or beta-gal control adenovirus.
The MOI is the ratio of the virus to cells (Infectious units/cells) and is calculated based on the viral titer and the number of cells plated. Optimal MOI can vary greatly by the target cell. Ideally, the lowest MOI that provides 100% infection should be used. An MOI that is too high will result in excessive CPE and an MOI that is too low will have reduced infection efficiency.
Adenovirus is able to infect any cell expressing the coxsackie-adenovirus receptor (CAR) on the host cell surface, which mediates viral entry. If the target cells don’t express CAR or express it at low levels, an additive such as the ViraDuctin™ Adenovirus Transduction Reagent can be used to increase transduction efficiency up to 12-fold. Gene expression can be analyzed 48-72 hours post-infection, depending on the gene and the amount of virus used for transduction.