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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-glutamine||PBS pH 7.4 without calcium or magnesium|
|Polybrene||Lenti-X 293T cells (or alternative cell lines)|
|Target cell line (any human cancer cell line)||Titered lentivirus containing your sequence of interest|
|6-well dishes||Selection antibiotic (e.g., Puromycin)|
|Microcentrifuge tubes||Pipette tips|
|Lentivirus volume (μL)||0||100||50||10||5||1|
|DMEM complete + 10 µg/mL polybrene volume (µL)||500||400||450||490||495||499|
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.
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.