Primary cells have a finite life span of less than 10-50 passages in vitro depending on cell type. Immortalizing your cell line will eliminate the frequent need to re-establish fresh cultures from specimen and will provide a consistent cellular material, capable of extended proliferation and possess similar or identical genotype and phenotype as the parental tissue, throughout the duration of your research experiment.
Immortalization Strategies for Mammalian Cells
Several methods exist for immortalizing mammalian cells in culture. With years of experience, abm has developed the most comprehensive cell immortalization products comprising of ready-to-use retroviral, lentiviral and adenoviral vectors.
Replicative senescence is an irreversible growth arrest triggered by a DNA damage signal from critically short telomeres, thus restoration of telomerase activity in primary cells can extend cell proliferation and achieve immortalization. Many studies have shown that co-expression of hTERT with other immortalizing agents (namely CDK4 and Bmi1, both of which silences tumor repressor p16) provides a more authentic normal cell model with well-defined genetic background.
Overriding the normal cell cycle control points is another way of bypassing cellular senescence, and the most commonly used and the best understood of this class of oncogenes is Simian Virus 40 large T antigen (SV40). SV40 Large T antigen binds to a number of proteins, but the most important are p53 and Rb which, when functioning normally, are negative regulators of cell division. Similarly, using siRNA to inactive p53 and/or Rb can also initiate unlimited cellular life span. Other oncogenes include c-Myc, Ras and HPV 16 E6/E7 and their introduction, likewise, can efficiently immortalize primary cells.
While most cell types can be established from the use of hTERT and other oncogene expression, B and T lymphocytes have proven refractory to immortalization to these immortalizing agents, and have only been shown to be immortalized through use of specific methods such as EBV.