Cell Immortalization Protocol Using Lentiviral Particles
Background
None of the primary cells can expand for several rounds of proliferation, which is known as the Hayflick limit, because each round of proliferation shortens the telomeres. When telomeres reach a critical shortening length, DNA damage is triggered, leading to cellular senescence. If you are trying to culture a population of rare primary cells, the incidence of immortalization can be increased by transfection techniques that introduce exogenous immortalization genes into the target cells or induce mutations in senescence-associated genes, thereby establishing a stable immortalized cell line.
Since many primary cells are difficult to transfect, the simplest and most efficient way to introduce gene mutations into primary cells is through viral infection. Immortalization of most cells can be achieved by stable expression of lentivirus in target cells with TERT or TAg. When TERT or TAg is introduced into the cells, the positive cells are screened with the help of fluorescent or resistance genes carried by the viral vector, and the cell morphology and proliferation are subsequently observed.
The hallmarks of successful immortalization are:
- The cell type is not transformed (analyzed by observation).
- The cells can divide consistently and stably (verified by simultaneous passaging analysis with primary cells).
Materials and Reagents
- Primary cell
- Cell immortalization lentiviral particles (Choose the appropriate lentivirus particles according to your experimental demands, e.g., SV40 small/large T antigen lentivirus (CMV, Puro), human TERT lentivirus (CMV, Puro), human CDK4 lentivirus (CMV, Puro), etc.)
- 6-well plate
- Antibiotics
- Water bath
- Incubator
Procedure
- Plate target primary cells in a 6-well plate at density of 0.5-2 x105 cells per well.
- Thaw the lentiviral particles in 37°C water bath and immediately remove it from the water bath once thawed.
- Transduce the cells in a 6-well plate with immortalizing lentivirus or GFP-lentivirus (positive control) at a MOI of 10-100. Dilute the lentivirus with the fresh complete medium if cytotoxicity is a problem.
- The next day, remove viral supernatant and add appropriate complete growth medium to the cells and incubate at 37°C.
- After 72 hours incubation, passage the transduced cells in complete medium supplemented with the appropriate amount of selection antibiotic.
- Pick antibiotic-resistant colonies and cultivate continuously in complete medium with antibiotics to test the immortalization.
* For research use only. Not intended for any clinical use.