Production of Lentiviral Vectrors with Calcium Phophate


Day 1:

  • Seed 5×106 293T cells in a 10 cm plate to achieve a confluency of about %80 after 24 h.

Day 2:

  • Two hours before transfection, replace the medium with 10 ml of fresh medium preheated at 37°C.
  • Prepare a mix of three vectors:
  • pLox-EWGFP: 21 µg
  • psPAX2: 21 µg
  • pMD2G: 10.5 µg
  • Add water up to 912 µl and mix
  • Add TE 1X 33 µl and mix
  • Add CaCl2 (2.5M) 105 µl and mix
  • Add 1050 µl of HBS2X, drop wise and slowly under agitation by vortexing
  • Vortex severely for 1 min
  • Incubate for 2-5 min, it should be cloudy now. Add drop-wise to the plate

(Note: buffers should be mixed and agitated before start.)

Day 3:

  • Remove medium around 14-16 h post-transfection (typically early in the morning) and put 6-7 ml/dish of fresh pre-heated medium.

Day 4:

  • Harvest the supernatant twice (morning and evening). Keep it at 4°C over the collecting period.

Day 5:

  • Harvest the supernatant in the morning and pool the collected supernatants, centrifuge 5 min at 1500 rpm to remove cell debris and pass through a 0.45 μm or 0.22 μm filter.
  • This supernatant contains virus particles and can be used for transduction, frozen at -70°C for future use, or to be concentrated.




  • NaCl 280mM
  • Hepes 100mM
  • Na2HPO4 1.5mM, 7.11≤pH≤7.13

TE 1X:

  • 10 mM Tris-Cl, pH 7.5
  • 1 mM EDTA


  • 2.5M in bi-distilled water

(Adapted from Trono Lab Protocols)



Low vector titers are often caused by inefficient transfection of 293T cells. Transfection efficiency can vary significantly and depends on several factors including the following. (1) When the 293T cells are plated for transfection, they must be trypsinized well to prevent clumping and allow formation of a uniform monolayer. Cells should be 50% to 70% confluent at the time of transfection. (2) Plasmid DNA of high purity and quality is crucial to achieve high-efficiency transfection. DNA concentrationtion and quality should be determined by spectrophotometric analysis and gel electrophoresis. Commercially available plasmid DNA extraction kits can be used to obtain highly purified supercoiled plasmid DNA. Prior to transfection, the DNA should be sterilized by ethanol precipitation. The air-dried pellet is then resuspended in sterile deionized, distilled

water. (3) The pH of the 2× HeBS solution is particularly critical and should be exactly 7.05. It is important to bubble the 2× HeBS solution with air as the DNA/CaCl2 solution is added dropwise. Once added to the cells, a fine precipitate

should develop that is readily visible under the microscope.

Vector titers are also influenced by the stability of the vector particles, which is affected by factors such as temperature, pH, freeze-and thaw frequency, and incubation conditions. VSV-G-pseudotyped HIV-1 vector particles have a half-life of 10.4 ± 1.2 hr at 37°C, 1 to 2 days at room temperature, and about 1 week at 4°C (Higashikawa and Chang, 2001). Changes in pH can dramatically affect vector stability. Although VSV-G-pseudotyped HIV-1 vectors are stable at pH 7.0, their half-life at pH 6.0 or 8.0 is <10 min (Higashikawa and Chang, 2001). In

order to prevent pH changes in vector supernatants, add HEPES buffer to the 293T medium at a final concentration of 10 mM.

(Ref: Curent Protocols in molecular biology-Unit 16.22)



Concentration of Lentiviral Vectors

  1. Place supernatants in sterile ultracentrifuge tubes
  2. Balance the weight of the tubes
  3. Spin at 40000 g for 2.5 hr at 4°C. (If using a fixed angle centrifuge, mark where the pellet should be as you often do not get a visible pellet.)
  4. Remove the tubes and discard the medium. (Transfer the centrifuge bucket to the hood and remove the tubes inside the hood). Virus pellet is loose, be careful not to disturb it. Leave a small amount of the medium to remain in the tube (about 1/100 of the primary volume)
  5. Suspend the pellet with the remaining medium.
  6. Let shake (~200 rpm) in the cold room or refrigerated shaker for 2-16 h.
  7. Collect media containing virus from the bottom of the tube and transfer to a tube
  8. Save 20 µl for titration of the viral stock and aliquot the rest and store at -70C.