Lentiviral Titer: for Gene Expression or Gene Editing

Authors: Abu Musa Md Talimur Reza

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This protocol describes the production of lentiviral particles by transfecting HEK293T cells with lentiviral plasmids using Lipofectamine 2000. The generated viral particles can then be used to efficiently deliver genetic material into target cells for gene expression or gene editing studies.

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Requirements list:

1. HEK293T cells (cultured and healthy)

2. Transfer plasmid (e.g., lentiCRISPRv2 hygro)

3. Packaging plasmid (psPAX2)

4. Envelope plasmid (pMD2.G)

5. Lipofectamine 2000 reagent

6. Opti-MEM Reduced Serum Medium

7. Dulbecco’s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS)

8. Antibiotics (e.g., Penicillin-Streptomycin)

9. Phosphate Buffered Saline (PBS), sterile

10. Polybrene (hexadimethrine bromide) — for enhancing viral transduction

11. Hygromycin B (selection antibiotic) — for selecting transduced cells

12. 0.45 µm syringe filters (for viral supernatant filtration)

13. 24-well Tissue culture plates (or other culture vessels as needed)

14. Pipettes and sterile pipette tips

15. Sterile Eppendorf tubes (for preparing transfection mixes)

16. Cell culture incubator (37°C, 5% CO₂)

17. Biosafety cabinet / laminar flow hood

18. Centrifuge (optional for clearing cell debris)

19. Sterile serological pipettes or pipettors

20. Sterile syringe

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Protocol:

Day 0: Cell Seeding

• Seed HEK293T cells in a 24-well plate to reach 70–80% confluency at transfection time

  
  

Day 1: Transfection

1. Remove medium from cells.

2. Wash cells once with 500 µL PBS, then aspirate.

3. Add 350 µL Opti-MEM per well.

4. Prepare transfection complexes (per 24-well plate):

   • Tube -DNA mix:

     • Take 75 µL Opti-MEM medium

     • Transfer plasmid (e.g., lentiCRISPRv2 hygro): 0.068 pmol for each well of a 24-well (1.64 pmol/10 cm)

     • Packaging plasmid (psPAX2): 0.054 pmol for each well of a 24-well (1.30 pmol/10 cm)

     • Envelope plasmid (pMD2.G): 0.03 pmol for each well of a 24-well (0.72 pmol/10 cm)

       
       

   • Tube - Lipofectamine 2000:

     • Take 75 µL Opti-MEM medium

     • 2.5 µL Lipofectamine 2000 - DNA: lipofectamine 2000 = 1 : 3 (may be used lesser or higher amount, a trial experiment with different ratios is recommended to identify what works best in your hand)

5. Incubate the Tubes for 5 min at room temperature.

6. Combine DNA mix to Lipofectamine mix, drop by drop with gently mix by pipetting.

7. Incubate the combined mix for 30 min at room temperature to allow complex formation.

8. Add this DNA-Lipofectamine 2000 complex (~150 µL) dropwise to each well of a 24-well plate.

9. Gently mix by swirling in a figure-8 and back-and-forth motion.

10. Incubate cells at 37 °C for 6 hours - check the cells health after 3 hours, if the health of the cells deteriorate then incubate 3-4 hours (3 hours minimum).

11. Remove the Opti-MEM containing complexes and discard.

12. Add 500 µL fresh medium with 10% FBS and 1 % penicillin/streptomycin

    
    

Day 3 (48 hours post-transfection): Virus Harvesting

1. Collect the medium containing virus.

2. Filter the collected supernatant through a 0.45 µm filter to remove cell debris.

3. Use the viral supernatant immediately for transduction of recipient cells or store at 4°C for short term.

4. Adjust volume with fresh medium with 10% FBS

5. Add polybrene (2–4 µg/mL medium) to the recipient cells during infection.

6. Optionally, collect viral supernatant again at 72 hours post-transfection for higher yield and repeat the above steps: 1- 5.

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DNA and Lipofectamine 2000 amount for different cell culture vessels:

Culture Vessel	Surface Area (cm²)	Volume Plating Media	DNA (µg)	Lipofectamine 2000 (µL)
96-well	0.3	100 µL	0.2	0.6
24-well	2	500 µL	0.8	2.4
12-well	4	1 mL	1.6	4.8
6-well	10	2 mL	3.2	9.6
60-mm dish	20	3 mL	6.4	19.2
10-cm dish	60	10 mL	20	60