VSVG Psudotyped Retrovirus Production   

Abstract

Materials and Reagents

1. Dulbecco's modified eagle medium (DMEM) (Life Technologies, Gibco^®, catalog number: 11995-065 )
   
2. Fetal bovine serum (FBS) (Gemini Bio-Products, catalog number: 900-108 )
   
3. Penicillin/streptomycin solution (Life Technologies, Gibco^®, catalog number: 15140-122 )
   
4. NaCl (Sigma-Aldrich, catalog number: S7653 )
   
5. HEPES (Sigma-Aldrich, catalog number: H7523 )
   
6. Na₂HPO₄ (Sigma-Aldrich, catalog number: S7907 )
   
7. CaCl₂ (Sigma-Aldrich, catalog number: C5080 )
   
8. Chloroquine (Sigma-Aldrich, catalog number: C6628 )
   
9. Sodium butyrate (Sigma-Aldrich, catalog number: B5887 )
   
10. Gag/pol (Cell Biolabs, catalog number: RV-111 )
    
11. VSVG and retrovirus vector (Clontech, catalog number: 631512 )
    
12. Calcium-phosphate (Ca-P) transfection solution (see Recipes)
    
13. 2x HeBS (pH 7.0) (see Recipes)
    
14. DMEM culture medium (see Recipes)
    

Equipment

1. 24-Well plate
   
2. Centrifuges
   
3. Water bath
   
4. 10 cm tissue culture plate
   
5. 0.45 micro filter
   

Procedure

1. Day 1
   Plate healthy 293 cells in 10 cm tissue culture plate (7 x 10⁶ cell/well in 10 ml culture medium).
   
   
2. Day 2
   1. Prepare for Ca-P (calcium-phosphate) transfection solution.
   2. Add 2x HeBS (pH 7.0) 450 μl to the Ca-P transfection solution and continually mix by vortexing.
   3. Replace cell medium with fresh DMEM culture medium containing 25 μM final concentration of chloroquine.
   4. Sprinkle the 900 μl retrovirus vector mixture from step on cells and incubate cells at 37 °C.
   5. After 3-4 h, replace cell medium with fresh DMEM culture medium, add sodium butyrate at final concentration of 10 μM and incubate at 37 °C.
   Note: Must be removed after 12-14 h.
   
   
3. Day 3
   Replace the DMEM culture medium containing sodium butyrate with fresh DMEM culture medium. Incubate cells at 32 °C O/N.
   
   
4. Day 4
   Harvest viral supernatant and store it at 4 °C. Then add fresh DMEM culture medium to the cells and continue to incubate cells at 32 °C.
   
   
5. Day 5
   Repeat day 4. Place healthy 293 cells for titering next day.
   
   
6. Day 6
   Repeat day 4. Combine all the supernatant together and filtered through 0.45 micron filter. (If high tittering virus is required, spin the supernatant at 25,000 RPM for 3 h at 4 °C. Remove supernatant carefully, and resuspend viral pellet in less than 0.5 ml of supernatant.)
   
   
7. Add 5 μl of concentrated virus to 293 cells (50% confluent) for tittering test. Snap-freeze by dry ice/ethanol concentrated virus and store at -80 °C.
   

Recipes

1. DMEM culture medium
   Supplemented with
   10% FBS
   1% Penicillin/streptomycin solution
   
2. 2x HeBS (pH 7.0)
   0.28 M final NaCl
   0.05 M final HEPES
   1.5 mM final Na₂HPO₄
   Adjusted to pH 7.0
   
3. Mix the following for Ca-P transfection solution
   54 μl 2 M CaCl₂
   15 μg gag/pol (if use package cell line which expresses gag/pol, no need to add it)
   6 μg VSVG
   9 μg retrovirus vector
   Use sterile water to make total volume to 450 μl and mix well.