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This protocol provides a method that allows the production of high titer lentivectors that preferentially transduce neurons. The lentiviral vectors produced using this protocol were used in previous studies, including re-introduction of CD38 gene expression into the hypothalamic neurons of CD38 knock-out mice.
(1) Human embryonic kidney (HEK) 293T cells
(2) Plasmids: lentiviral transfer vector
(3) Lentiviral packaging vectors: Packaging mix containing pLP1, pLP2 (pRev), pLP/VSVG
(4) Dulbecco's modified Eagle's medium (DMEM)
(5) Penicillin-streptomycin-glutamine (100x)
(6) Phosphate-buffered saline (PBS)
(7) FBS
(8) Polybrene
(9) 2.5 M CaCl2
(10) 2x HEPES-buffer (280 mM NaCl, 50 mM HEPES, 1.5 mM Na2HPO4 [pH7.05])
(1) 10-cm cell culture dishes
(2) 50 ml conical centrifuge tubes
(3) Steriflip-GP filter (0.22-µm) unit
(4) Ultracentrifuge tubes 17.0 ml
(5) Tissue culture incubator at 5% CO2
(6) SW28.1 rotor
(7) ultracentrifuge
(8) Fluorescent microscope
(9) Bio safety cabinet
(10) Centrifuge
Day 1: HEK cell seeding
1. Collect pre-confluent HEK 293T cells grown in DMEM supplemented with 10% FBS, 50 U/mL penicillin G and 50 µg/mL streptomycin (pH7.35).
2. Seed 1 × 106 HEK 293T cells in 10 cm dish in Step 1 and add 10 ml of DMEM supplemented with 10% FBS. Swirl the cells thoroughly to obtain even distribution across the surface of the dish. Incubate the cells for 24 h at 37°C.
Day 2: Transfection
3. Observe the dishes on Day 1. The cells should be approximately 60% confluent.
4. Exchange the culture medium for fresh medium (DMEM and 10% FBS, 10 ml) and further incubate the cells for 30 min in a CO2 incubator.
5. Mix 10 µg of packaging mix with 10 µg of lentiviral transfer vector in a 1.5 ml tube. Dilute the plasmid mix with filtered ddH2O to a total volume of 450 µl.
6. Add 50 µl of 2.5 M CaCl2 to the plasmid mix and then add 500 µl of 2xHEPES-buffer while vortexing.
7. Add all of the transfection mixture (spreading in drops) to the plate. Swirl the plates gently and incubate under 5% CO2, 37 °C overnight (16 h).
Day 3: Observe the cells and change the media
8. Observe the cells.
9. Remove media, wash cells twice with 5 ml of pre-warmed PBS (-), then add 9.5 ml of fresh DMEM and 10% FBS, and further incubate under 5% CO2, 37 °C overnight.
Day 4: Harvest and concentrate the viral particles
10. Collect supernatant from the dish 40 h after transfection.
11. Clear the supernatant of cell debris by filtering through a 0.22-µm filter.
12. Concentrate the viral particles by ultracentrifugation. Centrifuge the supernatant at 120,000 x g for 1.5 h at 4 °C using a swinging bucket rotor. We use a Beckman SW28.1 rotor (the capacity is 17 ml/tube, total 6 tubes): supernatant obtained from 2 dishes (18 ml) can be concentrated with one tube.
13. Pour off the supernatant. The pellet will be almost invisible.
14. Resuspend the viral pellet in 90 µl of PBS (-). The viral suspension can be used for both in vitro and in vivo experiments: however, if purer grade quality is needed, purify the virus through a sucrose cushion following the published protocol.
The titers of viral stocks were measured by transducing HeLa cells. Although the titration can be conducted after completing the viral concentration (Step 13), we usually start from Day 3 to shorten the whole process.
Day 3: HeLa cell seeding
15. Seed 1 × 105 HeLa cells on a twelve-well plate in 1 ml of DMEM and 10% FBS, add polybrene to a concentration of 6 µg/ml. Incubate the cells at 5% CO2, 37°C overnight.
Day 4: Infection of HeLa cells with lentiviral vectors
16. Make a ten-fold serial dilution of the lentivector preparation (from a dilution of 10-3 to 10-6) in PBS. The actual range to test will depend on the concentration of the viral preparation.
17. Add each viral dilution to HeLa cells growing in a monolayer as instructed in Step 16, mix thoroughly, but gently and incubate the cells at 37°C.
Day 5-8: Titration of lentiviral vectors
18. Grow the cells for another 3 days (total 88 h after infection). It takes more than 48 h before GFP fluorescence becomes visible: however, the period differs depending on strains of lentiviral vectors and cells used for titration.
19. Determine the percentage of labeled cells: if the marker is GFP, count GFP-expressing cells.
20. Calculate the biological titer (TU/ml, transducing units) as described previously.
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