Adenoviral vectors will transduce a wide range of target cells resulting in high-level gene expression. They are non-integrating vectors so expression in dividing cells is progressively lost. First generation vectors may be suitable for applications where transient, high level activity is sufficient, and where immune responses to the vector or transgene are acceptable. Adenoviral vectors can package Promoter-Transgene-Poly A cassettes ranging in sizes up to 6.0 kb.
The process of creating adenoviral vectors involves inserting the transgene into a small shuttle plasmid so that the transgene cassette can be excised with two restriction enzymes with almost unique cleavage sequences absent in most other DNA sequences. This cassette is then transferred to the adenovirus vector plasmid (pAdeno X, Clontech Laboratories or pAd5F35, The Journal of Gene Medicine Volume 6, Issue 6 , Pages 631 - 641). The adenovirus vector is released with Pac I and then 293 cells are transfected to make the final recombinant adenovirus vector.
In the VDL, all adenovirus vectors are produced in compliance with GLP standards.
Production of adenovirus vectors can begin at 1 of 3 steps. Service can be initiated at the cloning stage where we clone your transgene into the adenovirus backbone plasmid, at the transfection stage where you've done all of the cloning, or at the expansion stage where you provide us with a virus you want expanded and purified. Beginning at cloning or transfection requires more interaction between the VDL and you. Briefly, once cells have been transfected, an agar overlay is applied to the cells so that virus-producing cells will form plaques.
Several plaques are selected, expanded, HIRT DNA is analyzed, and virus lysates are sent to you for testing transgene expression. Once a plaque is selected for expansion, it undergoes a small expansion for production of a viral lysate. The viral lysate is used to infect a cell factory of 293 cells. The virus produced is purified by cesium chloride gradient ultracentrifugation. To see the procedure for producing adenovirus vectors in outline form see our Adenovirus Production Outline.
Quality assurance and quality control tests available for adenoviral vectors include titering, replication competent adenovirus detection and sterility and endotoxin testing.