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 temperat