Production of VSVG fake retrovirus

vector containing the blistered oral inflammatory virus (vesicularstomatitisvirusG, VSV-G) protein has been shown to deliver the
gene
efficiently to a variety of cells. Its efficiency is affected by the growth rate of relative cells and the level of phosphatidylserine on the cell membrane., Materials and
Reagents
1.DMEM（GIBCO,11995-065）
2.Fetal
Serum
FBS (GEMBIO900-108)
3.Penicillin/Streptomycin Solution (GIBCO15140-122)
4.NaCl（SIGMAS7653）
5.HEPES（SIGMAH7523）
6.Na
2
HPO
4
（SIGMAS7907）
7.CaCl
2
（SIGMAC5080）
8.Chloroquine, Chloroquine (SIGMAC6628)
9.Nabutyrate（SIGMAB5887）
10.Gag/pol（CellbiolabsRV-111）
11.VSVG and retrovirus vectors (Clontech631512) , equipment
1.24-well plate
2.Centrifuges
3.Water bath , experimental steps
1.Day 1: Place healthy 293 cells on a 10 cm
tissue
culture
plate. (10 ml
10
7×10
6
per
)
2.Day 2:
(1) Prepare Ca-P transflyction fluid.
(2) add 2×HeBS (pH7.0) 450 μl to Ca-P trans-dye and continue to vortex well.
(3) replace the cell culture solution with a fresh DMEM media containing the final concentration of 25
25
chloroquine.
(4) gradually sprinkled a mixture of 900 sl retrovirus vectors on the cells and incubated the cells at 37 degrees C.
(5) 3-4 h, replace the cell culture with a fresh DMEM media, add sodium butyrate at a final concentration of 10 m, and incubate the cells at 37 degrees C. Note: Must be removed after 12-14 h.
3.Day 3: Replace the DMEM media containing sodium butyrate with a fresh DMEM media and incubate the cells overnight at 32 degrees C.
4.Day 4: Harvest the virus and store it at 4 degrees C. A fresh DMEM medium is then added to the cell and continues to incubate the cell at 32 degrees C.
5.Day 5: Repeat Day 4. Place healthy 293 cells for use the next day.
6.Day 6: Repeat Day 4. Gather all the top clear and filter through the 0.45
filter
filter. (If a high-tittering virus is required, centrifuge 3 h at 4 degrees C, 25,000 RPM.) Carefully remove the upper clear and use less than 0.5 ml of rehanging virus particles).
7.Add 5 sl of concentrated virus to 293 cells (50% fusion) for tittering testing. Freeze the virus with dry ice or ethanol and store it at -80 degrees C.