Primary cells undergo a finite number of population doublings and reach a state called replicative senescence after limited cell divisions. GeneCopoeia’s Cell Immortalization Reagents use recombinant lentivirus carrying genes, such as the simian virus 40 (SV40) T antigen, the human telomerase reverse transcriptase protein (hTERT), CDK4, HOXB, HOXA, cMyc, Bmi1 and HPV-16 E6/E7 to induce immortalization and significantly prolong primary cells' lifespan.
GeneCopoeia Lentifect™ Lentiviral Particles are produced from a standardized protocol using purified plasmid DNA and the proprietary reagents EndoFectin™ Lenti (for transfection) and TiterBoost™ solution. The protocol uses a third-generation self-inactivating packaging system meeting BioSafety Level 2 requirements.
The Lentifect™ particles include CMV or EF1α promoter for efficient expression of the immortalization inducer gene in target cells with puromycin or hygromycin resistance marker for selection of stably transduced cells. The immortalization lentivirus particles without antibiotic selection marker are also available.
Provided as 4 vials of 25 µl of lentiviral particles with titers of ~1 x 108 TU/ml. Lentifect™ particles are shipped on dry ice and must be stored at –80°C immediately upon receipt. Avoid repeated freeze-thaw cycles as this will reduce titers.
The lentiviral expression construct was validated by full-length sequencing, restriction enzyme digestion and PCR-size validation using gene-specific and vector-specific primers. The product is confirmed free of bacteria, fungi and common Mycoplasma contamination.
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 ºC 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 ºC
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