General Guideline about Genes in Cell Immortalization

Primary cells closely represent the physiological state of a particular cell type in vivo, but they are susceptible to replicative senescence, so their value is limited in laboratory application. This is especially true when large number of cells are required for biochemical analysis, genetic manipulations or genetic screening. It is also a factor in the study of rare hereditary human diseases, since the biological samples collected (biopsies or blood) are usually small in size and contain limited number of cells. On the other hand, continuous cell lines are not obstructed by replicative senescence, but they often contain many genetic mutations, showing an unstable karyotype and protein expression patterns that are not compatible with the cell type they represent. Immortalized cells combine the physiological characteristics of primary cells and the long-term culture life of continuous cell lines, while avoiding the replicative senescence of the former and the unstable karyotype of the latter.

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. The most recently discovered 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.

• Nonmalignant
• Normal cell cycle controls, functional p53 and pRB checkpoints
• Contact inhibited
• Anchorage dependent
• Retain normal growth responses to serum and mitogens
• Require growth factors for proliferation
• Possess a normal karyotype
• Do not show changes associated with transformation such as tumorigenicity or growth in soft agar

In some cases, more than one immortalization agent may be required to successfully immortalize a particular cell type. For example primary cell lines may be immortalized using a combination of hTERT with one or more of the following: genes encoding viral (human papilloma virus-16 (HPV-16) E6/E7) or non-viral (CDK-4 and Bmi-1) oncoproteins.

General Guidelines for Cell Immortalization

Cell immortalization is a very complex process and the exact biological mechanisms are still not well understood. However, over the years of cell immortalization, scientists have observed:

• There are two main types of primary cells: one that can be cultured for 20-50 passages before aging; and ones that can only be passed fewer than 10 passages. Cells that survive a life span of 20-50 passages under in vitro culture conditions mainly include fibroblasts and retinoblasts. Cells with a life span of less than 10 passages are mostly epithelial cells, such as breast and ovarian epithelial cells.
• It is recommended that SV40 T antigens be used for primary cells that are difficult to immortalize, such as epithelial cells. In addition, if a more defined genetic background is required, combination of Rb or p53 siRNA and hTERT can be used.
• It has been shown that the introduction of hTERT may induce apoptosis in primary epithelial cells and other cells with a life span of less than 10 passages. It is recommended to use SV40 T antigens for these cells. In many epithelial cells, epithelia growth factor (EGF) has been shown to be able to extend their life span to 10-20 passages before senescence. Therefore, one may add some recombinant EGF (10 ng/mL) to expand cell life span before hTERT gene transduction.
• For some primary cell types, it has been demonstrated that overexpression of SV40 T antigen or hTERT alone is not sufficient for successful immortalization. However, a combinational expression of SV40 T antigen and hTERT or other genes have been shown to be effective.
• After transduction, drug selection is generally unnecessary for primary cells of less than 10 passenges, as the immortalization process will select for the clones capable of growing indefinitely.

General Procedure for Cell Immortalization

• Plate the target cells in one well of 6-well plate at density of 1-2 x 10⁵ cells/well.
• The next day, thaw the concentrated recombinant lentivirus in a 37°C water bath and remove it from the bath immediately when thawed.

  Note: After thawing, we recommend that the supernatant not be frozen again for future use since the virus-titer will decrease significantly.

• Infect the target cells in a 6-well plate with 4-20 µL/well supernatant in the presence of 4 µL TransPlus reagent.

  Note: TransPlus reagent is a polycation that neutralizes charge interactions to increase binding between the pseudoviral capsid and the cellular membrane.

• The next day, remove viral supernatant and add the appropriate complete growth medium to the cells and incubate at 37°C.
• After 72 hours incubation, subculture the cells into 2 x 100 mm dishes and add the appropriate amount of puromycin for stable cell-line generation.
• 10-15 days after selection, pick clones for expansion and screen for positive ones.

References

1. Hahn W.C. et al. Immortalization and transformation of human cells. Mol Cells, 2002, 13(3): 351-361.
2. Chiara M. et al. Cellular immortalization and neoplastic transformation. Cell Cycle, 2013, 12(11): 1804-1805.

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