Adenovirus Vector Information : Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. Homologous recombination in E. coli between shuttle plasmids and full-length adenovirus backbones (E1-deleted) is used for the generation of recombinant adenoviral vectors genomes. The adenovirus genomes are then analyzed to confirm their identity and integrity, and further linearized and transfected to generate a recombinant adenoviral vector in permissive human cells. These vectors are then purified by two sequential CsCl gradient centrifugations and subjected to a chromatography step in order to eliminate the CsCl and exchange buffers. Finally, the viral stock is characterized through the quantification of its viral particle content and its infectivity.
Adenovirus Vector Information
– Tropism: wide; preferent for CAR-expressing cells.
– Common Applications: short term in vivo studies.
– Procedure:
– Cloning in shuttle plasmid / homologous recombination.
– HEK-293 cells transfection.
– Amplification to 4x10E8 HEK-293 cells (standard production).
– Vector Purification: double cesium chloride gradient/ gel filtration chromatography.
– Characterization:
– Physical Particle (PP) quantification.
– Infectious particle (IU) quantification.
– Quality Control:
– Sterility tests: Tryptic Soy Broth and Fluid Thioglycollated Medium prepared according to the recommendations of the current European and United States Pharmacopeia.
– Mycoplasm test.
– RCA detection (under request).
– Average total yields (hAd): 4x10E12 PP, 3x10E11 IU
– Average titers (hAd): 1,5x10E12 PP/mL, 1x10E11 IU/mL
– Production time of AAV Vector (without cloning): 1,5 months.
The tropism of each Adenovirus serotype is indicated in the chart below.
Tissue |
Optimal Serotype |
CAR receptors |
Ad5 |
Macrophages, monocytes, intestinal cells |
Ad5/40 |
Neuronal origin cells |
Ad5/52 |
Neurons, respiratory cells |
CAV |