Editing AAV-Production-293T (section)

= <br>Large scale production of adeno-associated virus  =

1. Seed 1.2 Ã 10^7 293 cells in 150-mm dishes. 

2. Transfect 7.5 ÎŒg vector plasmid and 37.5 ÎŒg of the pRepCap-Helper plasmid using GeneExpresso Max 

3. After 72 h, crap the cells in the media and spin at 1000g. 

4. Suspend the pellet in a minimal amount of PBS (1 mL for each 150-mm dish of cells) and subject to 3â5 freeze-thaw cycles followed by sonication. 

5. Add sodium deoxycholate to 1.0% and 0.025% trypsin, incubate the lysate at 37°C for 30 min. 

6. Remove cellular debris by centrifugation (2000g). 

7. Purify virus by two CsCl ultracentrifugations. 

8. Add CsCl to the lysate to a final concentration of 1.41 gm/mL. 

9. Transfer the suspension to a Beckman centrifuge tube (cat. no. 344059) and centrifuged at 200,000g, using a SW-41 rotor in a Beckman L7/L8-M ultracentrifuge for 48 h. 

10. At the end of the spin, take out the tubes, collect 250 ÎŒL fractionations from the top of the tube. Pool the fractions corresponding to a density of 1.38â1.42 g/mL, and add fresh CsCl solution (1.41 g/mL in PBS), then repeat centrifugation. 

11. At the end of the second centrifugation collect and pool fractions corresponding to a density of 1.38â1.42. 

12. remove the CsCl by dialysing the pooled virus against four changes of PBS, or using a desalting column. Store the resulting AAV at â80°C until used.