Immortalizing Endothelial Cells via Retroviral Transduction of Viral Oncogenes

Abstract

Source: McAllister, S. C., Hanson, R. S., et al. An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus. J. Vis. Exp.(2017)

This video demonstrates retroviral transduction of primary endothelial cells with HPV16 E6/E7 oncogenes. The integrated oncogenes inactivate p53 and Rb tumor suppressors, extending cell lifespan. G418 selection enriches for transduced cells, yielding immortalized endothelial cultures suitable for transformation studies

Protocol

Transduction of endothelial cells (EC) with E6 and E7 Papillomavirus Genes

NOTE: We routinely use standard tissue culture flasks for growing primary EC. However, if unsatisfactory growth of EC are obtained then the use of commercial culture flasks should be considered.

1. Culture primary human dermal microvascular or lymphatic EC in a humidified incubator at 37 °C plus 5% CO₂ in EC growth medium (EGM; contains EC basal medium [EBM] supplemented with EGM-2 BulletKit [containing fetal bovine serum, hydrocortisone, human fibroblast growth factor-B, vascular EC growth factor, insulin-like growth factor-1, ascorbic acid, human epithelial growth factor, antibiotics, and heparin]) in a T75 cell culture flask to approximately 50% confluence.
2. For transduction, culture PA317 LXSN 16E6E7 cells in a humidified incubator at 37 °C plus 5% CO₂ in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum and antimicrobials in a T150 cell culture flask until they are approximately 90% confluent. Then incubate overnight in 16 mL of medium.
3. Clarify the PA317 medium by centrifuging at 300 × g for 5 min at room temperature.
4. Remove the EGM from the EC culture and overlay cells with 12 mL of the clarified PA317 medium and incubate for 4 h.
   NOTE: Centrifuging 16 mL of conditioned medium at 300 × g for 5 min results in a compact pellet that is not disturbed by the careful subsequent removal of 12 mL of clarified supernatant. However, if transfer of PA317 LXSN 16E6E7 cells to primary EC cultures is a concern (the packaging cell line will quickly outgrow EC), then filtration of the conditioned supernatant through a 0.45 µm filter before transfer can be performed.
5. Replace 6 mL of the PA317 medium with fresh EGM and incubate overnight.
6. Refeed EC with 12 mL fresh EGM and incubate a further 48 h.
7. To sub-culture the EC, remove medium and wash with 12 mL of phosphate-buffered saline (PBS) without cations, and add 3 mL of commercial enzymatic dissociation solution (e.g., TrypLE) and incubate at 37 °C for 3 min.
8. Transfer cell suspension to a 15 mL conical tube and centrifuge at 300 × g for 5 min at room temperature. Resuspend the resulting cell pellet in fresh EGM and divide evenly into 3 × T75 flasks with a final volume of 12 mL EGM per flask.
9. To select for EC transduced with E6 and E7, add G418 to a final concentration of 200 µg/mL for two passages, after which the transduced EC can be plated for infection or frozen in liquid nitrogen.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BCBL-1 cells	NIH AIDS Reagent Program	3233
PA317 cells	ATCC	CRL-2203
Neonatal dermal microvascular endothelial cells	Lonza	CC-2505
EBM-2 Basal Medium	Lonza	CC-3156
EGM-2 BulletKit	Lonza	CC-3162
RPMI
DMEM
PBS with calcium and magnesium