hTERT Cell Immortalization Reagents
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Product Details
Description
Several methods exist for immortalizing mammalian cells in culture. One such method is to use viral genes, for example 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 in culture, and the mechanism has been well documented. 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.
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 (e.g. human). 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 maintain a stable genotype and retain critical phenotypic markers.
However, over-expression of hTERT in some cell types,especially in primary epithelial cells, fails to induce cell immortalization and instead may induce cell death. Recent research has found that co-expression of hTERT catalytic subunit with either p53 or RB siRNA can immortalize human, primary, ovarian, epithelial cells, providing a more authentic normal cell model with well-defined genetic background (Yang G. et al., Carcinogenesis 2007; Yang G. et al., Oncogene 2007). Likewise, over expression of Ras or Myc T58A mutants has also been found to be able to immortalize some primary cell types (Sears R. et al., Genes & Dev. 2000).
With years of experience in cell immortalization, ABM has developed the most comprehensive cell immortalization products that comprise of plasmids and retroviral and adenoviral vectors for hTERT, p53 and RB, siRNAs, and SV40 T antigens. We also offer ready-to-use recombinant retroviruses and adenoviruses. All these tools will make your cell immortalization projects simpler and easier than ever before.
All our retroviral vectors are derived from Moloney Murine Leukemia Virus (MoMuLV).
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 (e.g. human). 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 maintain a stable genotype and retain critical phenotypic markers.
However, over-expression of hTERT in some cell types,especially in primary epithelial cells, fails to induce cell immortalization and instead may induce cell death. Recent research has found that co-expression of hTERT catalytic subunit with either p53 or RB siRNA can immortalize human, primary, ovarian, epithelial cells, providing a more authentic normal cell model with well-defined genetic background (Yang G. et al., Carcinogenesis 2007; Yang G. et al., Oncogene 2007). Likewise, over expression of Ras or Myc T58A mutants has also been found to be able to immortalize some primary cell types (Sears R. et al., Genes & Dev. 2000).
With years of experience in cell immortalization, ABM has developed the most comprehensive cell immortalization products that comprise of plasmids and retroviral and adenoviral vectors for hTERT, p53 and RB, siRNAs, and SV40 T antigens. We also offer ready-to-use recombinant retroviruses and adenoviruses. All these tools will make your cell immortalization projects simpler and easier than ever before.
All our retroviral vectors are derived from Moloney Murine Leukemia Virus (MoMuLV).
Cell Immortalization Guide 
Adenoviral Infection Protocol
Retroviral Infection Protocol
Notes
NOT FOR RESALE without prior written consent of ABM. This product is distributed for laboratory research only.
Caution: Not for diagnostic use.
FAQs
- pLenti-EF1α-hTERT-YFP, will it produce a fusion product? Can it be used to study translocation? Which filter do I have to use to see YFP?
- I would like to immortalize antigen specific mouse T cells to generate a clonal antigen specific T cell line. What would you suggest is the best method that you have experience with? How much would it be for you do accomplish this?
- Can your hTERT lentivirus immortalize other mammalian primary cells of non human origins?
- I work on marine mammal cell lines.... would this method work with seal and beluga primary kidney cell lines? I use them for virus isolation....Which method would you try first?
Do the usual antibiotics (penicillin and streptomycin) or a fungicide (amphotericin B) usually being added to the primary epithelial cell cultures interfere with the immortalization process via SV40 large T antigen (lower somehow the transfection efficiency)? Must we remove the above compounds before immortalization with a retroviral or a lentiviral vector?
Normally all the routine antibiotics do not interfere the infection efficiency of lenti/retro viral vectors.
pLenti-EF1α-hTERT-YFP, will it produce a fusion product? Can it be used to study translocation? Which filter do I have to use to see YFP?
hTert is linked to YFP by 2A. Since 2A has 'cleavage' activity by tricking the ribosomes to skip, hTERT and YFP will NOT be translated as a fusion product, but as separate proteins; As a result, the construct may not be suitable for studying translocation from cytoplasm to nucleus.
YFP’s peak excitation and emission wavelengths are 513 nm and 527 nm. Both GFP and YFP can be excited with a 488 nm blue laser.
YFP’s peak excitation and emission wavelengths are 513 nm and 527 nm. Both GFP and YFP can be excited with a 488 nm blue laser.
I would like to immortalize antigen specific mouse T cells to generate a clonal antigen specific T cell line. What would you suggest is the best method that you have experience with? How much would it be for you do accomplish this?
We have been able immortalize essentially any cell type including difficult to immortalize human hepatocytes and brain cells, but not T cells. We tried a couple of times before for other clients, but all have failed. The reason is that T cells are resistant to both transfection and lentiviral infection. So the immortalization gene can not get into the cells. We now learned that retrovirus can have limited transduction efficiency on T cells. So we can give it a try for you.
Can your hTERT lentivirus immortalize other mammalian primary cells of non human origins?
Yes, we have successfully used it on mouse and rat cells.
I work on marine mammal cell lines.... would this method work with seal and beluga primary kidney cell lines? I use them for virus isolation....Which method would you try first?
We have immortalized bee cells before and other lower species. I would recommend using Lenti-SV40 first. Additionally we do offer custom immortalization using all of our immortalization reagents we have. It is affordable and will not charge you if the project fails.