Production of neuron-preferential lentiviral vectors

doi:10.1038/nprot.2007.89

Method Article

Production of neuron-preferential lentiviral vectors

https://doi.org/10.1038/nprot.2007.89

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Genetics

lentivirus

gene transfer

Purkinje cell

Bergmann glia

cerebellum

Adenoviral vectors widely used to transfer foreign genes into neuronal cells possess tropism for glial cells ^{1, 2} 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 ^{3, 4}, including re-introduction of CD38 gene expression into the hypothalamic neurons of CD38 knock-out mice ⁵.

Human embryonic kidney (HEK) 293T cells (ATCC cat. no. CRL-11268) CRITICAL 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 (See Table 1)

Polybrene (Sigma cat. no. 10768-9)

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)

REAGENT SETUP

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 [pH 7.05])

Production and concentration of lentiviral vectors

Day 1: HEK cell seeding

Collect pre-confluent HEK 293T cells grown in DMEM supplemented with 10% FBS, 50 U/mL penicillin G and 50 µg/mL streptomycin (pH 7.35).
Seed 1 x 10⁶ HEK 293T cells in 10 cm dish in Step 1 and add 10 ml of DMEM supplemented with 10% FBS (Table 1). Swirl the cells thoroughly to obtain even distribution across the surface of the dish. Incubate the cells for 24 h at 37 ºC. CRITICAL 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 (Figure 1 and 2), the lot must be chosen by producing lentiviruses using various lots of FBS as exemplified in Table 1, then assessing the tropism for neurons upon viral injection to mouse or rat brains.

Day 2: Transfection

Observe the dishes on Day 1. The cells should be approximately 60% confluent. CRITICAL STEP More than 80% confluency at this point results in low pH of the culture medium at viral harvest.
Exchange the culture medium for fresh medium (DMEM + 10% FBS, 10 ml) and further incubate the cells for 30 min in a CO₂ incubator.
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.
Add 50 µl of 2.5 M CaCl₂ to the plasmid mix and then add 500 µl of 2xHEPES-buffer while vortexing.
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). CRITICAL STEP Incubation under 3% CO₂ was recommended in a previous protocol⁶. 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

Observe the cells. CRITICAL 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.
Remove media, wash cells twice with 5 ml of pre-warmed PBS(-), then add 9.5 ml of fresh DMEM + 10% FBS, and further incubate under 5% CO₂, 37 ºC overnight. Day 4: Harvest and concentrate the viral particles
Collect supernatant from the dish 40 h after transfection. CRITICAL 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.
Clear the supernatant of cell debris by filtering through a 0.22-µm filter.
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.
Pour off the supernatant. The pellet will be almost invisible.
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 ⁶. 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

Seed 1 x 105 HeLa cells on a twelve-well plate in 1 ml of DMEM + 10% FBS, add polybrene to a concentration of 6 µg/ml. Incubate the cells at 5% CO₂, 37 ºC overnight. Day 4: Infection of HeLa cells with lentiviral vectors
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.
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 ºC. Day 5-8: Titration of lentiviral vectors
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. CRITICAL 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.
Determine the percentage of labeled cells: if the marker is GFP, count GFP-expressing cells.
Calculate the biological titer (TU/ml, transducing units) as described previously ⁶.

4 days for procuction and concentration of lentiviral vectors

6 days for Titration of lentiviral vectors

Critical steps are included in "Procedure" section.

See Table 2.

As the tropism of lentiviral vectors for neurons depends substantially on the lot of FBS added to the culture medium (DMEM), transduced cell types and proportions are expected to differ depending on the lot of FBS used (Fig. 1). It should be noted that the tropism of lentiviruses is not related to their transduction efficiency in HEK 293T cells. Lentiviral vectors produced by adherence to this protocol should have titer of 1 x 10⁸ ~ 1 x 10¹⁰ TU/ml and if the lot of FBS is properly determined, >90% of the cells transduced in vivo are neurons in the cerebellum (Fig. 2).

Iino, M., et al. Glia-synapse interaction through Ca²⁺-permeable AMPA receptors in Bergmann glia. Science 292, 926-929 (2001).
Terashima, T., Miwa, A., Kanegae, Y., Saito, I. & Okado, H. Retrograde and anterograde labeling of cerebellar afferent projection by the injection of recombinant adenoviral vectors into the mouse cerebellar cortex. Anat Embryol (Berl) 196, 363-382 (1997).
Torashima, T., Okoyama, S., Nishizaki, T. & Hirai, H. In vivo transduction of murine cerebellar Purkinje cells by HIV-derived lentiviral vectors. Brain Res 1082, 11-22 (2006).
Torashima, T., Yamada, N., Itoh, M., Yamamoto, A. & Hirai, H. Exposure of lentiviral vectors to subneutral pH shifts the tropism from Purkinje cell to Bergmann glia. Eur J Neurosci (2006).
Jin, D., et al. CD38 is critical for social behavior by regulating oxytocin secretion. Nature (2007) in press.
"Tiscornia, G., Singer, O. & Verma, I.M. Production and purification of lentiviral vectors. Nat. Protocols 1, 241-245 (2006).":http://www.natureprotocols.com/2006/06/23/production_and_purification_of.php

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Production of neuron-preferential lentiviral vectors

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Introduction

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Production of neuron-preferential lentiviral vectors

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Figures

Introduction

Reagents

Equipment

Procedure

Timing

Critical Steps

Troubleshooting

Anticipated Results

References

Associated Publications

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