Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

1. Packaging of Lentiviral Vectors

Lentiviral vectors are produced by cotransfection of a lentiviral transfer vector and other plasmids required for packaging into 293T cells by calcium phosphate transfection method. We use 10 100-mm tissue culture dishes in this protocol. It can be scaled up or down depending on applications. The 293T cell line is maintained in Dulbecco’s modified Eagles medium (DMEM) with high glucose (4500 mg/L), supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin in 37 °C incubator with 5% CO₂.

1. Seed 293T cells at 30-40% confluence to 10 100-mm tissue culture dishes (3 x 10⁶ cells/dish) in culture medium. Return the cells to incubator.
2. After 20-24 h culture, check the cell density. The cells should be about 80% confluence at the time of transfection.
3. Prepare a 50-ml tube. Add 4.4 ml TE79/10 (1 mM TrisHCl, 0.1 mM EDTA, pH 7.9) minus the total volume of the following plasmid DNA. Add 100 μg lentiviral transfer plasmid (Figure 1), 58 μg pMDLg/pRRE, 31 μg pCMV-G, 25 μg pRSV-Rev, 600 μl 2M CaCl₂. Gently Mix.
4. Prepare another 50-ml tube. Add 5 ml 2x HBS (0.05 M HEPES, 0.28 M NaCl, 1.5 mM Na₂HPO₄, pH 7.12).
5. Take the DNA-CaCl₂ mixture by 10-ml pipette and add to the tube containing 2 x HBS, dropwise while vortexing the tube.
6. Keep the precipitation reaction at room temperature (RT) for 30 min.
7. Remove the culture dishes from incubator. Mix the precipitation reaction well by vortexing. Add 1 ml of suspension to each 100-mm dish containing cells. The suspension must be added slowly, dropwise while gently swirling the medium in the dish. Return these dishes to the incubator and leave for 5 h.
8. Remove the medium from the culture. Add 6 ml fresh culture medium containing 6 mM sodium butyrate to each dish. Return the cultures to incubator. After overnight culture, if there is a fluorescent reporter in the construct, check reporter gene expression under fluorescent microscope. Usually, over 80% of the cells express the reporter gene if it is driven by a ubiquitous promoter (e.g. CMV promoter).
9. Two days (40-44 h) after transfection, collect supernatant from 10 dishes into 2 50-ml tubes (about 30 ml each tube). Freeze the supernatant in -80 °C freezer or go to next step.

2. Concentration and Purification of the Vectors

1. Centrifuge the freshly collected or thawed supernatant at 900 g (about 2000 rpm) for 10 min to remove any cell debris in the supernatant.
2. Attach a 60-ml syringe to a 0.2-μm SFCA syringe filter. Transfer supernatant from 50-ml tube to the syringe. Filter the supernatant into a polyallomer centrifuge tube.
3. Take 5 ml 20% sucrose (prepared in PBS) up in a 5-ml pipette. Insert the pipette to the bottom of the centrifuge tube containing supernatant. Slowly add the sucrose solution under the vector supernatant. Repeat these steps for supernatant from another tube.
4. Centrifuge the supernatant at 11000 rpm and 4 °C for 4 h with Beckman SW28 swing rotor.
5. Remove the supernatant. Add 150 μl 4% Lactose (prepared in PBS) to each centrifuge tube. Resuspend the pellets.
6. Transfer the concentrated vector from all centrifuge tubes to a 1.5-ml tube. Leave the tube on ice for 15 min.
7. Mix the vector suspension by pipetting. Spin with microcentrifuge at full speed (about 16000 g) for 1 min.
8. Transfer supernatant to a new 1.5-ml tube. Divide the final sample into 20 μl aliquots and stored them in -80 °C freezer.

3. Titration of the Vectors

1. Seed 5 x 10⁴/well of HT1080 cells in 12-well plate in 1 ml DMEM medium supplemented with 10% FBS.
2. After overnight culture, count cells from one well and score the cell number.
3. Make 5-fold serial dilution (1:5, 1: 25; 1:125; and 1:625) of the concentrated vector with culture medium. Add I μl of each diluted vector to separate wells. The samples may be duplicated to increase accuracy.
4. Add 1 μl 4 mg/ml Polybrene (Hexadimethrine Bromide) in each well containing vector and into a well without vector. Mix by gently shaking the plate. Return to incubator for 48 h.
5. Remove medium from the cell culture wells. Wash each well with PBS. Add 250 μl 1x trypsine-EDTA solution to the cells. When the cells are detached (3-5 min), add 1 ml culture medium. Resuspend the cells by pipetting. Transfer cell suspension to 1.5 ml centrifuge tubes.
6. Centrifuge at 900 g for 6 min. For vectors with a fluorescent reporter gene (e.g. GFP), go to step 3.7 for FACS analysis. For vectors without a reporter, go to step 3.8 for real time qPCR.
7. For vectors containing a fluorescent reporter gene, remove the supernatant and resuspend the pellet with 300 μl of 3.7% formaldehyde in PBS. Determine the percentage of the reporter positive cells by FACS analysis. The titer will be represented as transduction units per milliliter concentrated vector (TU/ml).

For example, if 1 x 10⁵ cells was transduced with 1/25 μl (0.04 μl) vector and 30% cells are reporter positive, the titer will be:

Only use the dilutions fall in a linear relationship between the percentage of positive cells and the amount of vector added to calculate titer. The final titer should be an average of the titers obtained from transductions of at least 2 different amounts of the vector.

8. For vectors without a fluorescent reporter gene, extract genomic DNA from HT1080 cells using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. Amplify vector sequence in genomic DNA using ABI Prism 7000 Sequence Detection System (Applied Biosystems) with primers (in HIV-1 PBS/psi region ¹⁷) 5′-CCGTTGTCAGGCAACGTG-3′ and 5′-AGCTGACAGGTGGTGGCAAT-3′, and TaqMan probe 5′-FAM-AGCTCTCTCGACGCAGGACTCGGC-TAMRA-3′. Albumin gene that is a single copy gene in the genome (2 copies/cell) was also amplified with primers 5′-TGAAACATACGTTCCCAAAGAGTTT-3′ and 5′-CTCTCCTTCTCAGAAAGTGTGCATAT-3′, and probe 5′-FAM-TGCTGAAACATTCACCTTCCATGCAGA-TAMRA-3′ as an internal control. Determine the copy numbers of vector and albumin by PCR in 96-well plate according to the manufacture’s instruction with the following program: 50 °C for 2 min, 95 °C for 10 min, and 35 cycles of 95 °C for 15 sec and 60 °C for 2 min. Ten-fold serial dilutions of plasmids of known concentration (represented as copy number) containing the template sequences should also be amplified to create a standard curve for quantification of unknown samples. The titer will be represented as integration units per milliliter concentrated vector (IU/ml).

4. Transduction of Neocortical Cultures

Neocortical cultures containing both neurons and glia are prepared from mouse cortices using a two-step plating procedure as previously described ¹⁸. Neocortices obtained from fetal mice at 14-16 days gestation are plated onto a previously established glial monolayer in MEM supplemented with 10% FBS, 20 mM glucose and 2 mM glutamine in 24-well tissue culture plate.

1. After 5 days in vitro, add 10 μM cytosine arabinoside (Ara-C) in neocortical culture to inhibit non-neuronal cell division. Continue culture the cells for 2 days.
2. Warm culture medium in 37 °C water bath for 5-10 min. Replace Ara-C containing medium with fresh culture medium (500 μl/well).
3. Add vector with desired MOI (multiplicity of infection; the ratio of the number of vector particles to the number of target cells) to the culture. Continue culture for 24 h. We use MOI of 1-10 (usually 5) in primary cortical cultures.
4. Replace the culture medium with fresh medium. Continue culture. If there is a reporter gene in the vector construct, check cells under fluorescent microscope 2 days after transduction. Reporter gene expression will be visible in neurons 2-7 after transduction, depending on the vector design and the dose used.

5. Representative Results

The titers of lentiviral vectors produced with this protocol range 10⁸-10¹⁰ IU/ml, which are suitable for transduction of a variety of cell types from CNS both in vitro and in vivo. Table 1 and figure 2 show a representative result using the vectors produced by this protocol. We transduced murine neocortical cultures with lentiviral vectors expressing green fluorescent protein (GFP) controlled by synapsin (SYN) promoter or glial fibrillary acidic protein (GFAP) promoter. Seven days after transduction, we performed immunostaining to label neurons and astrocytes with anti-NeuN and anti-GFAP antibodies, respectively. As shown in table 1 and Fig. 2A, after transduction with the vector carrying the synapsin promoter, over 90% of neurons (NeuN⁺ cells) express GFP and no astrocytes (GFAP⁺ cells) express this reporter gene. When GFAP promoter is used in the vector construct (Fig. 2B), about 80% of astrocytes (GFAP⁺ cells) express GFP; all GFP⁺ cells are astrocytes as confirmed by colocalization with GFAP and the absence of GFP expression in NeuN-labeled cells. These results demonstrate that lentiviral vectors are very efficient to deliver transgenes to cells from CNS and cell-specific gene expression can be achieved when appropriate promoters are used.

Figure 1. Schematic representation of HIV-based lentiviral vectors and the packaging plasmids. The HIV-1 provirus is shown at the top. The elements for vector production are separated into four different plasmids. The lentiviral transfer plasmid contains a hybrid 5′ LTR in which the U3 region is replaced with the cytomegalovirus (CMV) promoter, the packaging signal (ψ), the RRE sequence, the central polypurine tract (cPPT), a gene of interest (e.g. a fluorescent reporter) along with a promoter of choice, and the 3′ LTR in which the cis regulatory sequences are completely removed from the U3 region. pMDLg/pRRE contains the gag and pol genes and RRE sequence from HIV-1 under the control of the CMV promoter. pRSV-Rev contains the coding sequence of Rev driven by the RSV promoter. pCMV-G contains the VSV-G protein gene under the control of the CMV promoter. PA indicates the polyadenylation signal from human β-globin gene.

Figure 2. Expression of reporter genes in mouse neocortical mixed culture transduced with lentiviral vectors carrying cell type-specific promoters. The cultures were transduced with LV-SYN-GFP (A) or LV-GFAP-GFP vectors (B) at a MOI of 5. Seven days after transduction, the cells were immunostained with anti-NeuN or anti-GFAP antibody. Upper panels show GFP fluorescence, middle panels show immunostaining and lower panels are merged images (GFP: green; NeuN or GFAP: red).

Table 1. Comparison of GFP expression in murine neocortical cultures transduced with lentiviral vectors carrying different promoters^a.
^aMurine neocortical cultures (5 x 10⁵/well in 24-well plate) were transduced with LV-SYN-GFP or LV-GFAP-GFP at an MOI of 5. Seven days after transduction, cultures were fixed and immunostained for NeuN or GFAP. The number of GFP and NeuN/GFAP expressing cells were counted in images from 10 fields per experimental condition. The values represent the percentage of neurons (NeuN⁺ cells) or astrocytes (GFAP⁺ cells) that also expressed the GFP reporter gene. The values shown are means ± SD from three independent experiments.