Adeno-associated virus (AAV vector) - Production Protocol, GuidelinesAAV protocol Download
AAV Production Protocol--Genemedi
1. AAV plasmid construction
The gene of interest is cloned into one of the ITR/MCS-containing AAV vectors to generate AAV-GOI. The purity and RNAse contaminants of AAV viral plasmid should be taken into consideration.
2. AAV packaging
The recombinant AAV viral plasmid AAV-GOI is co-transfected into the AAV-293 cells with AAV helper plasmid pHelper (carrying adenovirus-derived genes) and AAV-RC (carrying AAV-2 replication and capsid genes), which together supply all the trans-acting factors required for AAV replication and packaging in the AAV-293 cells.
3. Harvesting AAV particles
Collecting the cells and freeze/thaw the cell pellet to release the AAV virus.
4. AAV purification
Purify the AAV virus with gradient ultracentrifugation to separate contaminants from the impure AAV preparations.
5.AAV titer detection
Determine AAV titer by real-time quantitative PCR (QPCR) using primers targeting the AAV ITR. Titer values are then identified by comparison to a standard curve of a plasmid sample of known concentration.
6.Quality control of AAV
After AAV virus titer detection, the infection activity needs to be evaluated before animal experiments by infecting cells such as 293T, CHO to test the gene expression. MOI for AAV mediated gene transduction in cells will be controlled ranging from 10^4 to 10^5.
Adeno-associated virus (AAV vector) - Guidelines
|NIH Guidelines for Adeno-Associated (AAV) Lentivirus and Adenovirus||Guidance for Gene Therapy Clinical trials-FDA|
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