Lentiviral

Generating a stable cell line with lentiviral particles

Day 1:

- T-25 flask of packaging cell line HEK 293T cells is prepared for the next day (~70% confluent next day).

Day 2:

- T-25 flask of HEK 293T cells (70% confluent = ~2.5*10⁶) for each condition (passaged the previous day) is co-transfected with the transfection mix, including pMD2g (envelope), psPAX2 (packaging) and lentiviral (transfer*) plasmids in a 1:3:4 ratio, respectively (*pHR-, pLVX- or pLL7-based plasmids were used as transfer plasmids).

Co-transfection:
optiMEM : 300 µL
X-tremeGENE 9 transfection reagent : 25µL
pMD2g: 1.2μg 
pPAX2: 2.9μg 
Vector of interest: 4μg

- Mix is vortexed for a few seconds (3 s).
- Mix is incubated in room temperature (RT) for 15-30 minutes.

- Mix is added to 4 mL of the medium present in each flask.

Day 4:

- 48 h after transfection, lentiviral particles are filtrated from the supernatant of the culture by passing it through 0.45 μm filter using a syringe.
- 1 mL-2 mL of each supernatant is added to target cells (RPE1 - 50% confluent) in a 6-well plate (transduction).

- Transduction is done in the medium without serum + protamine (8 ug/mL).
- The remaining supernatant should be kept in the fridge (+4).

Day 5:

- After being transduced for 24 h, infected RPE1 cells are passaged (in T-25 flask if possible).

- Cells are brought back to the lab after 3 passages in a new flask (T-25).

- Subsequently, RPE1 cells are selected by fluorescence-activated cell sorting (FACS) according to the fluorescence level of transduced protein.

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