TITLE: Production of Neuron-Preferential Lentiviral Vectors Protocol
AUTHORS: creative-biogene

[DESCRIPTION]
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 ofCD38gene expression into the hypothalamic neurons of CD38 knock-out mice.

[STEPS]
SECTION: Experiment Summary
1. 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.
3. 
SECTION: Main Reagents
2. (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])
SECTION: Equipment
3. (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
SECTION: Production and concentration of lentiviral vectors
4. 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 CO2incubator.
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 CaCl2to 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 bothin vitroandin vivoexperiments: however, if purer grade quality is needed, purify the virus through a sucrose cushion following the published protocol.