Productionofneuronpreferentiallentiviralvectors

实验概要

Adenoviral vectors widely used to transfer foreign genes into neuronal cells possess tropism for glial cells and are toxic to infected cells. Alternatively, the use of lentiviral vectors for transducing neuronal cells has been prevailing because, in addition to their limited toxicity to infected cells, lentiviral vectors facilitate long-term expression of the transgene. The glycoprotein of lentiviral vectors that is critical for viral tropism is pseudotyped with that of vesicular stomatitis viruses (VSV-G). As the receptor for VSV-G is phosphatidylserine, it is generally thought that lentiviral vectors infect most of cell types: however, we have sometimes noted drastically different results in cell types transduced by nearly identical lentiviral vectors produced by slightly modified protocols. We found that pH of the culture media at viral harvest and lot variations in fetal bovine serum (FBS) preparations added to the culture media critically influence the resultant viral tropism. Based on our observation, 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.

主要试剂

Human embryonic kidney (HEK) 293T cells (ATCC cat. no. CRL-11268) sCRITICAL Although a lentiviral kit from Invitrogen (cat. no. K4975-00) supplies HEK 293FT cells, use of HEK 293T cells is recommended, as the lentiviral tropism for neurons is dependent on medium pH at viral harvest: HEK 293FT cells grow more rapidly than HEK 293T cells and therefore, pH of the culture medium in which HEK 293FT cells are growing decreases more quickly.
Plasmids: lentiviral transfer vector (Invitrogen cat. no. K4950-00 or St. Jude Children’s Research Hospital)
Lentiviral packaging vectors: Packaging mix containing pLP1, pLP2 (pRev), pLP/VSVG (Invitrogen cat. no. K4975-00)
Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, cat. no. D6546)
Penicillin-streptomycin-glutamine (100x) (Invitrogen cat. no. 10378-016)
Phosphate-buffered saline [PBS (-)] (Invitrogen cat. no. 10010-023)
FBS
Polybrene (Sigma cat. no. 10768-9)
CaCl₂ solution (2.5 M CaCl₂): 36.75 g of CaCl₂ in 70 ml double distilled (dd) H₂O. Adjust the solution to final volume of 100ml followed by filtering through a 0.22 µm filter.
2x HEPES-buffer (280 mM NaCl, 50 mM HEPES, 1.5 mM Na₂HPO₄ [pH7.05])

主要设备

10-cm cell culture dishes (Falcon cat. no. 353003)
50 ml conical centrifuge tubes (Falcon cat. no. 352070)
Steriflip-GP filter (0.22-µm) unit (Millipore cat. no. SCGP00525)
Ultracentrifuge tubes 17.0 ml (Beckman Coulter cat. no. 344061)
Tissue culture incubator at 5% CO₂
Beckman SW28.1 rotor (or equivalent)
Beckman ultracentrifuge (or equivalent)
Fluorescent microscope
Bio safety cabinet (SANYO cat. no. MHE-130AB3)
Centrifuge (Tomy cat. no. LC-120)

实验步骤

Production and concentration of lentiviral vectors
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 ºC.
sCRITICAL STEP Lots of FBS added to DMEM have a critical impact on the tropism of the resultant lentiviruses for neurons. As the lot of FBS that yields lentiviruses with high neural tropism is not related to transfection efficiency, the lot must be chosen by producing lentiviruses using various lots of FBS as exemplified, then assessing the tropism for neurons upon viral injection to mouse or rat brains.
Day 2: Transfection
3. Observe the dishes on Day 1. The cells should be approximately 60% confluent.
sCRITICAL STEP More than 80% confluency at this point results in low pH of the culture medium at viral harvest.
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 ºC overnight (16 h).
sCRITICAL STEP Incubation under 3% CO₂ was recommended in a previous protocol. However, incubation under 5% CO₂ does not cause any problem as transfection efficiency is not substantially influenced by medium pH when HEPES buffer, instead of BBS, is used for transfection.
Day 3: Observe the cells and change the media
8. Observe the cells.
sCRITICAL STEP The cells should not be reaching confluency at this point and should still have room to undergo 1-2 cell divisions, because the medium pH rapidly decreases after the cells reach confluency.
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 ºC overnight.
Day 4: Harvest and concentrate the viral particles
10. Collect supernatant from the dish 40 h after transfection.
sCRITICAL STEP The color of the culture medium (actually the color of phenol red) should still be red (pH 7.20 or higher), as the medium pH at this point is critical for tropism of the viruses being cultured. Therefore, do not use cells from the second harvest, as the medium pH substantially decreases after the first harvest. To obtain lentiviral vectors that preferentially transduce glial-cells, use viruses obtained 2 or 3 days after transfection: in such cases, change culture medium every day after transfection. In addition, it is preferable to use a lot of FBS that yields lentiviruses possessing tropism for glia, in combination with HEK 293FT cells.
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 ºC 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.
Titration of lentiviral vectors
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, 37ºC 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 37ºC.
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.
sCRITICAL STEP Titers should be determined using a volume of vector preparation that yields linear, dose-dependent transduction of target cells with a level not in excess of 20%. HeLa cells grow more slowly than HEK cells.
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.