| Steps Performed By |
Adenovirus Production Outline |
| Investigator |
- 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
|
| Investigator |
- 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
|
