Gene expression systems for mammalian cells have evolved significantly over the past decades, enabling targeted expression of complementary DNA (cDNA) in cultured cells. These systems primarily fall into two categories: DNA transient transfection vectors and viral expression vectors. Four major viral vectors have been developed: adenovirus, adeno-associated virus (AAV), lentivirus, and retrovirus. While adenovirus and AAV efficiently infect host cells in free form and express high levels of exogenous proteins, retroviruses, and lentiviruses integrate into the host cell genome. These integrating vectors do not cause cytotoxicity and can be used for stable transfection of mammalian cells, making them valuable tools for establishing stable cell lines. This article provides a comprehensive protocol for lentivirus production, cell infection, and selection of stable cell lines.

Figure 1. Stable lentiviral cell line construction.

Materials and Reagents Required

PEG8000 (5×) Preparation

1. Combine 8.766 g NaCl and 50 g PEG8000
2. Dissolve in Milli-Q purified water to a final volume of 200 mL
3. Sterilize by autoclaving
4. Store at 4°C

Protocol for Lentivirus Production

Day 1: Cell Preparation

1. Seed 293T cells in 100 mm culture dishes at a density that will yield 80-90% confluence by the next day
   • Critical: Use 293T cells in optimal condition, preferably plated the previous evening to ensure they are in logarithmic growth phase for transfection

Day 2: Transfection

1. Prepare Solution A: Mix plasmids in 250 μL Opti-MEM per 100 mm dish according to the following ratios (adjust based on your specific packaging system):
   • Shuttle plasmid (containing transgene): 20 μg
   • Packaging plasmid(s): 15 μg total (for third-generation systems, divide appropriately between the two packaging plasmids)
   • Envelope plasmid (VSV-G): 6 μg
2. Prepare Solution B: Dilute PEI (1 μg/μL) in 250 μL Opti-MEM. Use 2-3× the total plasmid mass (i.e., 82-123 μL PEI for 41 μg total plasmid)
3. Incubate both solutions separately at room temperature for 5 minutes
4. Combine the solutions by adding Solution B dropwise to Solution A while gently vortexing
5. Incubate the mixture at room temperature for 15-20 minutes to allow complex formation
   • Critical: Do not disturb the tube after this incubation period
6. Add the 500 μL transfection mixture dropwise to the 293T cells
7. Replace the medium with fresh complete medium after 4-6 hours or the following morning (option to perform half medium exchange: replace 50% of the medium)

Day 4: Virus Collection (48 hours post-transfection)

1. Perform all steps on ice or at 4°C
2. Pre-cool the centrifuge
3. Transfer supernatant to 15 mL centrifuge tubes
4. Centrifuge at 3000 rpm, 4°C for 10 minutes to remove cell debris
5. Collect supernatant using a syringe and filter through a 0.45 μm filter into a 50 mL centrifuge tube
6. Virus concentration: a. Add 1/4 volume of cold 5× PEG8000 solution to the filtered viral supernatant b. Immediately mix by inverting the tube c. Incubate on ice, inverting 5 times every 30 minutes, for a total of 5 inversions d. Store at 4°C overnight
• Note: Successful precipitation is indicated by the appearance of a white precipitate

Day 5: Virus Concentration and Target Cell Preparation

1. Pre-cool the centrifuge
2. Centrifuge the virus/PEG mixture at 4000g, 4°C for 20 minutes
3. In a biosafety cabinet, carefully remove the supernatant
4. Allow the tube to stand for 1 minute and remove any remaining supernatant
5. Resuspend the viral pellet in 200-400 μL cold PBS
6. Aliquot the concentrated virus into two 1.5 mL microcentrifuge tubes for two infection cycles
7. Store at -80°C or proceed immediately to infection
8. Seed target cells in 35 mm dishes or 6-well plates at approximately 70% confluence

Day 6: Target Cell Infection

1. Thaw viral stock on ice or at room temperature
   • Critical: Avoid repeated freeze-thaw cycles as they significantly reduce viral titer
2. Add polybrene to the viral suspension to achieve a final concentration of 8 μg/mL after addition to the culture medium
   • Note: Some cells are sensitive to polybrene; consider substituting with protamine sulfate if necessary
3. Add the virus/polybrene mixture to target cells and gently rock to distribute evenly
4. Return cells to the incubator
5. Monitor cell condition post-infection; when cells reach confluence, subculture them
6. When cells return to the logarithmic growth phase, a second infection cycle can be performed using the reserved viral aliquot

Protocol for Stable Cell Line Selection

Determination of Optimal Selection Antibiotic Concentration

1. Day 1: Seed target cells in a 6-well or 24-well plate to achieve 80-90% confluence the next day
2. Day 2: Add selection antibiotic at various concentrations (e.g., for puromycin, test 1-10 μg/mL in 1 μg/mL increments)
3. Day 3-7: Monitor cells daily and replace medium with fresh antibiotic-containing medium every two days
4. Determine the minimum concentration causing complete cell death within 3-5 days

Mixed Clone Selection

1. Day 1: Seed infected cells and non-infected control cells to achieve 70-80% confluence the next day
2. Day 2: Add the optimal antibiotic concentration determined previously
3. Continue selection for at least 48 hours; uninfected control cells should die completely
• Note: Ideally, 50-70% of infected cells should survive. Too few survivors may indicate excessive cell death, while too many survivors may result in low-expressing cells being retained
4. Day 5: Reduce antibiotic concentration to 1/2-1/4 of the selection concentration for maintenance
• Critical: Selection is most effective when cells are actively dividing; puromycin selection requires at least 48 hours, with effective selection typically occurring over 3-10 days

Cell Banking and Validation

1. During expansion, cryopreserve cells regularly, ensuring at least 6 vials per cell line
2. Thaw one vial after 2-3 weeks to assess cell viability and growth characteristics
3. Validate transgene expression by appropriate methods (PCR, Western blot, fluorescence microscopy for fluorescent proteins, etc.)

Single-Clone Selection (Optional)

While mixed populations are often sufficient for many experiments, single-clone selection provides more homogeneous expression and greater stability over multiple passages.

1. Digest and dilute the mixed stable population to achieve single cells per well in 96-well plates (using limiting dilution or flow cytometry sorting)
2. Mark wells containing single cells
3. Allow cells to expand under selection pressure
4. For fluorescent marker-containing lentiviruses, ensure 100% of cells display fluorescence
5. Continue to expand promising clones
6. Validate expression by PCR or Western blot
7. Cryopreserve validated single clones

Troubleshooting Guide

1. Transgene Not Detected After Infection

Potential causes and solutions:

• Low viral titer: Test transfection efficiency using a GFP-expressing control vector; observe fluorescence in 293T cells 48h post-transfection. If fluorescence is abundant, transfection is likely successful.
• Defective plasmids: Verify plasmid quality and ensure they are endotoxin-free.
• Cytotoxicity: Transgene overexpression may be toxic to 293T cells; consider using an inducible expression system.
• Inappropriate promoter: The promoter may have low activity in target cells; test alternative promoters with higher activity in your specific cell type.
• Incompatible envelope protein: Research which envelope proteins work best with your target cells and consider changing the envelope plasmid.

2. Low Viral Titer

Standard unconcentrated viral titers should exceed 1×10⁷ TU/mL, with concentrated virus reaching 10⁸-10⁹ TU/mL. Potential causes and solutions:

• Insert size: Verify the transgene does not exceed the maximum capacity of the lentiviral vector.
• Cell density: Ensure optimal 293T cell density for transfection.
• Plasmid quality: Use high-quality, endotoxin-free plasmid preparations.
• Serum batch variation: FBS source and batch significantly affect transfection efficiency; consider testing different serum batches.

3. 293T Cell Detachment During Virus Production

Potential causes and solutions:

• VSV-G cytotoxicity: VSV-G envelope protein can cause cell fusion and detachment, particularly on day 3. Minimize disturbance and seed 293T cells at higher density.
• Transgene toxicity: If the transgene is toxic to 293T cells, consider using a drug-inducible lentiviral vector system.

4. Successful Infection of 293T Cells but Not Target Cells

Potential causes and solutions:

• Promoter activity: Test with a GFP-expressing vector to assess infection efficiency. If GFP expression is low, the promoter may have poor activity in target cells; identify a promoter with better activity in your cell type.
• Envelope protein tropism: Research and test alternative envelope proteins that work better with your target cells. Note that non-VSV-G pseudotyped lentiviruses are less stable during ultracentrifugation; use prolonged low-speed centrifugation instead.

5. Using Lentiviral Shuttle Plasmids for Transient Transfection

While not recommended, some lentiviral vectors (particularly third-generation systems) can be used for transient transfection. However, the LTR structure may affect transgene expression. It is generally preferable to use vectors specifically designed for transient expression.

6. MOI Calculation and Optimization

Multiplicity of Infection (MOI) refers to the ratio of viral particles to target cells. Different cell types require different MOIs for optimal infection efficiency.

MOI Calculation Formula

Volume of virus (μL) = (MOI × Number of cells) / (Viral titer [TU/mL]) × 1000

Example:

To infect 2×10⁵ cells at MOI=30 with a virus of titer 2.4×10⁸ TU/mL

Volume (μL) = (30 × 2×10⁵) / (2.4×10⁸) × 1000

= (6×10⁶) / (2.4×10⁸) × 1000

= 0.025 × 1000 = 25 μL

While higher MOI generally increases transduction efficiency, excessive MOI may cause cytotoxicity or result in multiple viral integrations per cell. It is advisable to test a range of MOIs to determine the optimal value for each target cell type.

Quick reference: Common Cell Lines MOI