Production of Retroviral Supernatant Using Transient Transfection of Producer Cells

The method given is modified from that of ref. 8.

1. Plate 7 x 106 Bosc23 cells/100-mm tissue culture dish the day prior to transfection. Cells are grown in a humidified 37°C incubator with 5% CO2.

2. On the day of transfection: Refeed Bosc23 cells with 8 mL GM + 10 pL of 25 mM chloroquine (see Note 17).

3. In a 5 mL tube, mix 35-40 pg of retroviral plasmid DNA with water to give a final volume of 875 pL.

4. Add 125 pL 2 M CaCl2and mix well.

5. Add 1 mL of 2X BES solution to the DNA/ CaCl2 mixture dropwise with bubbling or vortexing.

6. Gently add the DNA mixture to the Bosc23 cells.

7. Return the cells to the 37°C incubator at 5% CO2 for 6-12 h.

8. Aspirate the media and refeed with 27 mL of fresh GM.

9. Twenty-four hours after the start of transfection, refeed the Bosc cells with 9 mL of GM and transfer to a humidified 32°C incubator at 5% CO2 (see Note 18).

10. Forty-eight hours after the start of transfection, collect the supernatant and filter through a 0.45-pm filter. Aliquot the supernatant to cryogenic vials (50 pL and 1 mL) and rapidly freeze using dry ice or liquid N2. Store in a liquid nitrogen cryogenic tank.

11. Refeed the dish with 9 mL of GM and keep at 32°C.

12. Sixty hours after the start of transfection, collect the supernatant again. Filter, aliquot, and freeze as above.

13. Refeed the dish with 9 mL of GM and keep at 32°C.

14. At 72 h after the start of transfection, collect the supernatant for the last time (see Note 19). Filter, aliquot, and freeze as before.

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