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1. Transfection of Plasmids for Production of the Rev-dependent Lentiviral Particles
Set up: The Rev-dependent GFP lentiviral vector, pNL-GFP-RRE-SA, was described previously4,5,6. To assemble into a viral particle, the plasmid was contransfected into HEK293 T cells with an HIV-1 packaging construct, pCMVΔ8.2 (kindly provided by Dr. Dider Trono), and a plasmid carrying the VSV-G glycoprotein (pHCMV-G). The transfection was carried out using the calcium phosphate method.
Preparation of buffers: 10 X HBS (Hepes Buffered Saline) was prepared by dissolving 5 g of Hepes, 8 g of NaCl, 0.37 g of KCl, 1 g of Dextrose, 0.103 g of Na2HPO4 (anhydrous) in 100 mL of H2O. Aliquot the10 X HBS buffer into 1 mL aliquots and store at -20°C. At the time of transfection, convert the 10 X HBS to 2 X HBS with H2O (1: 5 dilution), and adjust the pH to between 7.05 - 7.12. (for 10 mL 2 X HBS, it usually requires approximately 50 μL of 1M NaOH). Filter sterilize the 2X HBS buffer by passing through a 0.22 μM Millipore filter. CaCl2 buffer was made by dissolving CaCl2 in 10 mM Hepes to a final concentration 2M. Adjust pH to 5.8, filter sterilize the buffer and store at 4°C.
Procedure:
- One day prior to transfection, seed 2 x 106 HEK293T cells in a 100 mm Petri-dish, and grow cells overnight in 10 mL DMEM + 10% FBS at 37°C, 5% CO2.
- The next day, remove supernatant from cells and replace with 5 mL fresh DMEM + 10% FBS, continuously culture cells for 4 hours.
- In a 5 mL polystyrene tube, add 480 μL 2X HBS buffer.
- In a second 5 mL polystyrene tube, add 60 μL 2M CaCl2 buffer, the plasmid DNA, and transfection TE buffer (1 mM Tris, 25 mM EDTA, pH 8.0) to a total volume of 480 μL. For each Petri-dish, plasmids should be added in the ratio of 10 μg of pNL-GFP-RRE-SA, 7.5 μg of pCMVΔR8.3, and 2.5 mg of pHCMV-G.
- Add the plasmid DNA-CaCl2 mixture dropwise to the 2 X HBS tube, and incubate at room temperature for 30 minutes. A fine precipitate will form.
- Add the 960 μL of the mixture dropwise by pipette to the cells. Incubate at 37°C, 5% CO2 overnight.
- The next day, remove the supernatant and add 10 mL fresh DMEM + 10% FBS, and continuously incubate the cells at 37°C, 5% CO2 overnight.
- At 48 hours post transfection, harvest the virus by removing the supernatant and transferring it into 50 mL sterile tubes. Add fresh 10 mL fresh DMEM + 10% FBS into each Petri-dish and continue to culture overnight. Store the virus supernatant at 4°C.
- Continue to harvest at 72 hours post transfection by collecting the supernatant. Combine all the supernatants, and centrifuge the supernatants at 500 x g for 15 minutes to pellet and remove cell debris.
- The supernatant were collected and filtered through a 0.22 μM Millipore filter. The virus was further concentrated through size-exclusion columns.
2. Concentrate Viral Particles and Determine Viral Titer
- Viral particles were concentrated by a size-exclusion column (100,000 MW cut off, Centricoin). Viral supernatant was loaded into the column, and centrifuged at 6,000 x g at 4°C for 15 minutes.
- Concentrated viral supernatant was collected, aliquoted, and stored at -80°C.
- The viral titer was determined by infection of a TNF-treated, HIV-1 positive Jacket cell line, J1.1 7. Cells were cultured in a 96 well plate at 2.5 x 105 cells per mL (100 mL total volume), and infected with 100 mL of serially diluted vNL-GFP-RRE-SA for a week. The titer (TCID50) of the particle was estimated by counting the GFP positive wells following the method of Reed and Muench 8.
3. Marking HIV-1 Positive Cells with the Lentiviral Particle, vNL-GFP-RRE-SA
Set up: a human CD4 T cell line, CEM-SS, was first infected with HIV-1. At 48 hours post infection, cells were superinfected with the lentiviral particles, vNL-GFP-RRE-SA. Following superinfection for another two days, GFP-positive cells were analyzed by flow cytometry. As a control, HIV-1 uninfected CEM-SS cells were identically infected with vNL-GFP-RRE-SA. Only the HIV-1-infected CEM-SS cells gave rise to GFP positive cells but not the HIV-1 uninfected CEM-SS cells.
Procedure:
- Two hours before infection, add polybrane to a final concentration 4 mg / mL. Count cells and take 2 x 105 cells for each infection. Infect cells with 200 ng (p24) of HIV-1NL4-3 for 2 hours.
- Washing cells by centrifuge at 300 x g for 10 minutes, resuspend cells into 2 x 105 cells / mL, and culture at 37°C for 48 hours.
- Count cells and take 2 x 105 cells per infection with vNL-GFP-RRE-SA. Add 100 mL of vNL-GFP-RRE-SA and incubate at 37°C overnight. Wash cells and resuspend into 2 x 105 cells / mL.
- Perform flow cytometry analysis at 48 hours post superinfection. Infected cells were pelleted and resuspended into 500 mL 1% paraformaldehede, incubated at room temperature for 20 minutes, and then analyzed on a FACScan analyzer (Becton Dickenson).
4. Representative Results
If the experiments are successfully performed, a sizable GFP population will be detected in HIV-1-infected CEM-SS cells by the flow cytometer, whereas, in the control, GFP positive cells will not be detected in HIV-1 uninfected CEM-SS cells.
If the experiments are successfully performed, the Rev-dependent lentiviral vector, vNL-GFP-RRE-SA, will permit the highly specific detection of replicating HIV in living cell populations, through measurement of green fluorescence protein (GFP) expression. In this example, harvested CEM-SS cells were stained with a PE-labeled rat monoclonal antibody against mouse CD24, HSA, and then analyzed on a flow cytometer for both HSA and GFP expression.
Figure 1. As shown here, a sizable GFP population was detected in HIV-1-infected cells superinfected with vNL-GFP-RRE-SA (Figure 1c). In contrast, GFP positive cells were not detected in either HIV-1 uninfected CEM-SS cells without lentiviral superinfection (Figure 1a) or HIV-1 uninfected cells with lentiviral superinfection (Figure 1b).