Lentiviral vectors allow you to create a clonal cell line overexpressing a transgene of interest. Such cell line can be developed following this 3-step protocol, validated on HEK293T cells and HeLa cells.
The first step consists of transducing the original cell line with the lentiviral vector expressing your transgene of interest. Depending on your objective, different transduction conditions can be considered:
For transduction protocols, see TechNote How to transduced cells with lentiviral vectors
Cells are grown for at least 72 hours to recover from transduction.
Cells are then collected and diluted at very low concentration to be seeded in 96-well plates. To ensure that a colony derives from a single transduced cell, we recommend to seed at 0.1 cell per well (eg: 1 cell/mL, 100µL per well).
Cells are grown for several days in 96-well plates, medium being renewed every 3 days.
Cell growth is regularly monitored by microscopy observation; colonies should appear within 1-2 weeks. Confirm through microscopy observation that the growing population derives from a single cell. Discard wells where several cells have been seeded.
Once the population have grown enough, pursue the amplification in larger wells (eg: 6-well plates) and proceed characterization of clones:
Depending on your purpose, select the clone(s) that display expected features regarding transgene expression level and transgene integration site(s).