As primary cells reach replicative senescence after a limited number of population doublings, researchers frequently need to re-establish fresh cultures from explanted tissue - a tedious process which can also add significant variation from one preparation to another. In order to have consistent material throughout a research project, primary cells with an extended replicative capacity are needed, or immortalized cells. The ideal immortalized cells are cells that are not only capable of extended proliferation, but also possess similar or identical genotype and phenotype to their parental tissue.
Several methods exist for immortalizing mammalian cells in culture conditions. One method is to use viral genes, such as the simian virus 40 (SV40) T antigen, to induce immortalization. SV40 T antigen has been shown to be the simplest and most reliable agent for the immortalization of many different cell types and the mechanism of SV40 T antigen in cell immortalization is relatively well understood. For the most part, viral genes achieve immortalization by inactivating the tumor suppressor genes (p53, Rb, and others) that can induce a replicative senescent state in cells. Recent studies have also shown that SV40 T antigen can induce Telomerase activity in the infected cells.
Another popular approach to cell immortalization is through the expression of Telomerase Reverse Transcriptase protein (TERT), particularly for cells that are most affected by telomere length, such as human cells. This protein is inactive in most somatic cells, but when hTERT is exogenously expressed, the cells are able to maintain sufficient telomere lengths to avoid replicative senescence. Analysis of several telomerase immortalized cell lines has verified that the cells immortalized by hTERT over expression maintain a stable genotype and retain critical phenotypic markers.
Recent studies have found that co-expression of the hTERT catalytic subunit with either p53 or RB siRNA can immortalize human primary ovarian epithelial cells, providing more authentic and normal cell model with well-defined genetic background. Likewise, overexpression of Ras or Myc T58A mutants have also been found to be able to immortalize some primary cell types. For the most part, viral genes achieve immortalization by inactivating the tumor suppressor genes (p53, Rb, and others) that can induce a replicative senescent state in cells.
The HOX genes (HOXB8, HOXA9, and HOXA10) are a family of homeodomain-containing transcription factors associated with malignancies such as acute myeloid leukemia and acute lymphoid leukemia. Accordingly, overexpression of the HOX genes can be used to immortalize various hematopoietic cells, including macrophages, hematopoietic progenitor cells, and myeloid progenitor cells.
BioCat offers a large number of recombinant lenti-, retro- or adenoviral particles for the expression of e.g. SV40 T antigen or hTERT to facilitate your cell immortalization project:
Tumor Suppressor Gene Inactivation
Please also see our comprehensive selection of ready-to-use immortalized cell lines.