The adeno-associated virus (AAV) is one of the most promising viral vectors for human gene therapy. This protocol, we outline a procedure based on transient plasmid cotransfection and CsCl isopycnic ultracentrifugation. This method can be used to generate high quality vector stock of any AAV variant. The experimental process of recombinant AAV production is as follows.
Day 1: Cell seeding
The day previous to transfection, prepare 15cm plates by seeding each platess with HEK-293. Be sure cells should be at 70%~80% confluence the next day.
Day 2: Transfection with plasmid mix.
Observe the plates seeded on Day 1. When cells are 70~80% confluent they are ready for transfection. Before transfection, remove the medium from the cells and replace with fresh medium. At the same time, transfections can be performed using polyethylenimine (PEI).
Day 3: Observe the cells and change the media
Remove the medium from the cells and replace with 15 ml of fresh DMEM + 2% FBS to each dish and incubate in 5% CO2, 37 °C overnight.
Day 5: Collect cell and supernatant
● Collect supernatant and place in tubes at 4 ℃.
● Using a cell scraper, scrape the transfected HEK-293 cells from each 15cm plate and place in centrifuge tubes at 4 ℃.

Virus may be stored at 4 °C for short periods (hours to days), but should be frozen at -20 °C or -80 °C for long-term storage. To reduce the number of freeze/thaw cycles, aliquot large-scale virus preps to smaller storage tubes prior to long-term storage.

BrainVTA could offer the high-quality adeno-associated virus (AAV) and satisfactory customized AAV services to researches.

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