Editing AAV-Production-293T (section)

= Purification of adeno-associated virus by heparin affinity chromatography  =

<br>1. Attach a 1-mL HiTrap heparin column to a 30-mL syringe with a concentric luerlok. 

2. Equilibrate column with 15 mL 1X PBS-MK 

3. Load fraction containing AAV to cloumn using a 5-mL syringe. Collect the effluent and pass it through the same column again. 

4. Wash column with 15â20 mL 1X PBS-MK. 

5. Elute virus from the column by applying 6â7 mL 1X PBS-MK/1 M NaCl. 

6. Dilute eluent with 1X PBS-MK to 15 mL. 

8. Concentrate virus by ultrafiltration (to approximately 1 mL). Repeat dilution and spin, then spin to 150â300 ÎŒL. 

9. This will yield 5 Ã 10^11 to 2 Ã 10^12 viral particles (titer is as high as 1 Ã 10^13 viral particles/mL). 

10. Aliquot the virus and store at â80°C. 

Production of recombinant adeno-associated virus by CaCl2 Transfection 

1. Split 293T cells into 10 15-cm dishes at a cell density of between 8â9.5 Ã 10^6 cells/dish, so that the cultures are about 70% confluent the following day. 

2. On the day of the transfection, prepare the following transfection mixture in a 50-mL Falcon tube (Nunc) by combining 700 ÎŒg pAAV and ppRepCapH at an equimolar ratio, 1.25 mL 2.5 M CaCl2, and make up to 12.5 mL with ddH2O. 

3. Add 12.5 mL 2X HBS (room temperature) at a steady slow rate. 

4. Incubate the transfection mixture for 1 min at room temperature, beginning from the addition of the 2X HBS. 

5. Transfer the entire 25-mL transfection mix to 200 mL of prewarmed growth medium (37°C/8% CO2) 

6. Mix gently by inversion. 

7. Remove culture medium from 15-cm dishes 

8. Add 22 mL of the transfection mix to each plate. 

9. Incubate at 37°C/8% CO2 for 2 to 3 d. 

10. Remove ~ 20 ml culture medium, leaving 2â3 mL in each plate. 

11.Scrap cells, pool cells and pellet cells by centrifugation at 1000g for 10 min. 

12. Resuspend cell pellet in 15-mL cell lysis buffer. 

13. Lyse cells by four cycles of freeze/thaw by alternatively placing in ethanol/dry ice and then a 37°C water bath. 

14. Add 3 ÎŒL of benzonase to the 15-mL lysate and vortexed briefly, then incubate at 37°C for 1 h. 

15. Centrifuge at 6500 rpm (18°C, 5000g) for 20 min to clarify the cell lysate. 

16. Store the clarified lysate at â80°C. <br>