Editing AAV-Production-293T (section)

= <br>Production of recombinant adeno-associated virus by CaCl2 Transfection: Protocol 3<br>  =

1. Day 1: 24 h prior to transfection, seed 15-cm tissue culture plates with 2.0â2.2 Ã 10^7 293 cells per plate in 25 mL DMEM. The plates should be approx 70% confluent at the time of transfection. 

2. Day 2: 2â3 h before the transfection, remove the culture media and replace with 25 mL IMDM per plate, prewarmed to 37°C. 

3. Allow the 2X HeBS buffer, 2.5 M CaCl2 and distilled H2O to equilibrate to room temperature. 

4. In Nunc 50-mL tubes, prepare the transfection mix. For each 15-cm dish, use 66 ÎŒg helper plasmid (pRepCapHelper), 11 ÎŒg AAV plasmid, 330 ÎŒL 2.5 M CaCl2 and make up to a final volume of 2.75 mL with distilled water. 

5. Filter the transfection mix through a 0.2-ÎŒm Acrodisc filter to sterilize and remove particles that may bind to the calcium phosphate precipitate. Dispense the solution into 2.5-mL aliquots in sterile disposable polystyrene tubes. 

6. Take an aliquot of transfection mix and while vortexing the solution vigorously, quickly add 2.5 mL of 2X HeBs buffer with a pipet gun and keep vortexing for a further 5â10 s. Leave the solution to stand for a further 50 s (total of approx 1 min for precipitate to form). 

7. Take a plate out of the incubator and quickly, but gently, add the 5-mL transfection solution dropwise in a circular motion around the plate, then swirl gently to mix, and return plate to the incubator. 

8. 8â15 h after transfection, aspirate the medium and replace with 25 mL of prewarmed DMEM. 

9. 48â60 h after transfection aspirate the media from the cells and discard. 

10. Add 25 mL prewarmed PBS to the plate, aspirate, and discard. 

11. Add 25 mL of PBS to each plate and detach cells using a cell scraper. Transfer the contents to 50-mL Nunc tissue culture tubes. 

12. Pellet cells at 200g for approx 5 min. 

13. Remove supernatant and resuspend cell pellets in 150 mM NaCl, 20 mM Tris-HCl pH 8.0. Volume to use is 10 mL per plate. 

14. Pool the cell suspension and split into two 25-mL aliquots in 50-mL Nunc tubes. 

15. Add sodium deoxycholate to a final concentration of 0.5% (1.25 mL of a 10% stock solution to each tube containing 25 mL). 

16. Add Benzonase to a final concentration of 50 U/mL (4.25 ÎŒL of 300 U/ÎŒL to each tube). 

17. Incubate in a 37°C water bath for 30â40 min, before removing cell debris by centrifugation at 3000g Ã 15 min, 4°C. 

18. Transfer the supernatant into another Nunc tube and heat in a 56°C water bath for 15 min. 

19. Freeze supernatant in a dry ice/ethanol bath. 

20. Thaw the supernatant and remove any cell debris by centrifugation at 3000g Ã 15 min, 4°C. 

21. Pool the two 25-mL aliquots to give a total of 50 mL from the five plates. Filter through a 32-mm 0.45-ÎŒm Acrodisc syringe filter to remove any particulate matter<br> 

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