Adenovirus Production Outline

Steps Performed By Adenovirus Production Outline
  • Obtains pShuttle plasmid from the Gene Vector Core
  • Clones gene of interest into pShuttle
  • Transforms plasmid and selects for Kanamycin-resistant colonies
  • Screens mini-prep DNA from colonies by restriction digests
  • Sends at least 100ug of the plasmid to the Gene Vector Core
Gene Vector Core
  • Expression cassette excised from pShuttle
  • Expression cassette ligated into the Adeno Viral DNA
  • Ligation reaction digested with Swa I and transformed into Stbl2 cells
  • Ampicillin-resistant colonies screened by restriction digests
  • Maxi-prep of the final plasmid are made
  • Adenoviral DNA digested with Pac I
  • HEK 293 cells transfected and monitored for plaque formation
  • Plaques picked and small-scale expansion performed
  • Positive samples sent to investigators to test and select sample for final expansion & purification
Gene Vector Core
  • Medium-scale expansion of investigator's selected sample for production of lysate
  • Viral lysate used to infect cell factory
  • Large-scale expansion harvested and purified on two CsCl gradients
  • Smaple desalted, aliquoted and frozen
  • QA/QC testing
  • Virus shipped by the Gene Vector Core to the investigator