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Generation of Stable Cell Lines Using Lentivirus

Lentiviruses are used widely to generate stable expression mammalian cell lines. They are used for gene down-regulation (by using shRNA) or for gene up-regulation (by using ORF of the gene of interest). This protocol can be used to generate stable cell lines expressing a gene of interest from an integrated lentiviral vector. Unlike the short term protein expression observed using transient transfection methods, generating cell lines using lentiviral vectors enables long-term protein expression studies. In addition, repeating experiments in a stable cell line as opposed to transiently-transfected cells increases reproducibility, since it eliminates the variation associated with repeated transient transfection.

Materials and Reagents

DMEM complete medium with 10% FBS and 4mM L-alanyl-L-glutaminePBS pH 7.4 without calcium or magnesium
PolybreneLenti-X 293T cells (or alternative cell lines)
Target cell line (any human cancer cell line)Titered lentivirus containing your sequence of interest
6-well dishesSelection antibiotic (e.g., Puromycin)
Microcentrifuge tubesPipette tips


  1. Firstly, determine the optimal dose of selective reagent for your target cell line. Thus, treat target cells with a range of doses of antibiotic and determine the lowest dose that kills all of the cells.
  2. Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting 20 µL of 10 mg/mL polybrene into 20 mL media.
  3. Rapidly thaw the lentiviral aliquot at 37 °C. Prepare a range of dilutions of the lentivirus in DMEM complete + 10 µg/mL polybrene. Mix the dilutions well.
  4. Dilution ratio01:51:101:501:1001:500
    Lentivirus volume (μL)0100501051
    DMEM complete + 10 µg/mL polybrene volume (µL)500400450490495499
  5. Add 0.5 mL of a single viral dilution to each well of the 6-well dishes.
  6. Make the cell solution by using the polybrene-containing media. Prepare a batch of cells as follows: Dilute 350,000 cells into a total volume of 7 mL of DMEM complete + 10 µg/mL polybrene. Mix well by inverting the tube.
  7. Perform "reverse transduction" by aliquot 1 mL of cell suspension (i.e., 50,000 cells) into each well of the 6-well dish.
  8. Note: Transducing too many cells relative to the number of virus particles reduces the transduction efficiency, leading to massive cell death upon antibiotic selection. The number of cells transduced should be enough that they can grow out in a reasonable amount of time, but not so many that they vastly outnumber the virus particles.

  9. Incubate the cells with the virus for 48-72 hours. Afterwards, gently aspirate the media from the cells.
  10. Add 1.5 mL DMEM complete containing the appropriate antibiotic. This is the beginning of the selection process, which will begin the selection of a stable cell pool.
  11. Observe the dish every day to ensure that the cells in the untransduced well are dying. Perform regular fluid changes and monitor the growth of the cells.
  12. Note: Depending on the efficiency of your transduction, you will see different degrees of cell death upon antibiotic selection. Cell death by some cells in the culture may adversely affect the surviving cells in the culture, thus it is important to do regular fluid changes and maintain optimal growth conditions for the surviving cells. Moreover, to achieve a stable cell pool, the antibiotic selection should last at least as long as it takes the untransduced cells to completely die.

  13. As polyclonal populations of resistant cells start coming through and the individual wells become confluent, expand into larger vessels. This selection method results in a polyclonal cell population, meaning that the transgene has integrated into different locations in the various cells in the culture. This is because the lentiviral integration is random.
  14. Once the polyclonal populations are growing well and have been sufficiently expanded, prepare cell stocks or harvest to test for protein expression.

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