Lentiviral vectors allow you to create a clonal cell line overexpressing a transgene of interest. Such cell line can be developed following the proposed 3-step protocol, validated on 293T cells and HeLa cells.
Step 1: cell transduction
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:
- High vector dose: use high vector dose to reach near 100% of transduced cells. This approach favors selection of clonal populations with several integrations of vector genome and high levels of transgene expression.
- Low vector dose: use low vector dose to achieve 30% of transduced cells. This approach favors clonal populations with single integration of vector genome and variable levels of transgene expression for each cell, due to position effect (variegation). Note that in this case, the proportion of clonal populations obtained is lower.
For transduction protocols, see TechNote How to transduced cells with lentiviral vectors
Step 2: Isolation of clonal populations
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).
Step 3: Amplification and selection of clonal populations
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:
- Transgene expression analysis : the method depends on your transgene, eg: direct fluorescence observation, ELISA, Western blot, RT-PCR (note: be aware that the transgene sequence from the vector genome may interfere with the reaction and give false positive results)
- Transgene integration sites: the number of integrated copies of vector genome can be determined by Southern Blot or qPCR; the integration locus can be determined by inverse PCR.
Depending on your purpose, select the clone(s) that display expected features regarding transgene expression level and transgene integration site(s).