I established the stable cell lines to express the protein driven by CMV promoter. It seems CMV promoter is easy to lose the expression of transgenic gene after several times cell passages. Does anyone suggest which promoter is the best to establish stable cell lines in colon cancer cell lines?
Hi,
with all necessary respect - you should look for a proper supervisor.
The loss of transgene expression is a normal phenomenon in the context of stable-cell-line generation (it is also depending from the nature of the transgene). But the CMV promoter is for sure not the reason why you loose it's expression - at least not in colorectal cancer cell lines. CMV works here especially well. It may help if you carefully look for the resistance gene encoded by your plasmid used for transfection, establish the necessary concentrations (of G418, zeocin, puromycin or what it is) for cell line selection and use it later on at reduced doses.
Regards
Michael
Thank for your reply. Colon cancer cell lines can robustly methylated promoter. I think that it is one of the reason that transgene lose the expression. another reason may be the clonal selection. the protein expressed by the transgene may be too toxic for the cells. this promoter has been used to many other types cancer cell lines in our lab and we never face any problem. Only colon cancer cell lines have this issue. we use the lentiviral vector to integrate the transgene to the genome.
Ok,
which cell lines exactly are you using?
Not all CRC lines are "CpG-Island methylators" ...
Ok, they are microsatellite instable AND have the CpG island methylator phenotype, too.
Bad choice then. Caco-2, SW480 or HT29 should be better then.
Dear Fang, i am having a similar problem as you have faced. Is there any best vector to use for HCT116 cells that you would suggest? Thanks in advance.
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