Immortalizing Endothelial Cells via Retroviral Transduction of Viral Oncogenes

Take a medium containing retroviral particles encoding viral oncogenes and an antibiotic-resistance gene.

Centrifuge the medium to remove cell debris.

Collect the supernatant containing the viral particles and overlay it on a culture of endothelial cells.

Incubate to allow viral entry.

The viral RNA is reverse transcribed into DNA, which integrates into the host genome.

The antibiotic-resistance gene produces a resistance protein. The oncogenes produce proteins that bind to tumor suppressor proteins that control cell division and inactivate them, extending cell lifespan.

Replace the medium with a fresh medium.

Incubate again to ensure stable viral DNA integration.

Remove the medium and wash the cells.

Add an enzymatic dissociation solution to detach the cells.

Transfer the cell suspension, centrifuge, and remove the supernatant.

Resuspend the pellet and transfer it into a flask.

Add an antibiotic to the medium.

Only cells expressing the antibiotic-resistance protein survive, selecting for cells immortalized by viral oncogene proteins.

Following an overnight incubation, collect the medium from the PA317 cell culture and clarify it by centrifugation at 300 times g for five minutes at room temperature. Replace the EGM medium from 50%confluent endothelial cell culture with 12 milliliters of the clarified PA317 medium. Incubate the cells for four hours.

Then replace six milliliters of the PA317 medium with six milliliters of fresh EGM medium and incubate the cells overnight. Afterward, remove the medium from endothelial cell culture and feed the cells with 12 milliliters of fresh EGM. Continue to incubate the cells for an additional 48 hours.

To subculture endothelial cells, remove the medium and wash the cells with 12 milliliters of cation-free PBS. Then add three milliliters of a commercial enzymatic dissociation solution and incubate the cells at 37 degrees Celsius for three minutes. Transfer the cell suspension to a 15-milliliter conical tube and centrifuge the cells at 300 times g for five minutes at room temperature.

Once pelleted, resuspend the cells in fresh EGM medium and divide the suspension evenly into three T75 flasks. Adjust the volume of the cultures to 12 milliliters of EGM per flask. Then add 200 micrograms per milliliter of G418 to select transduced endothelial cells.