So I have made several stable melanoma cell lines expressing a protein of interest using retroviral vector. The cells survive antibiotic selection while those without the construct are all dead. The cells expressing the phenotype that cell without the protein don't have.
However, when I try to do western blot to see if my protein level actually increase, I can't see any difference between the control and the supposedly overexpressing cell line. The efficiency is actually quite good as many of the transfected cells survive however.
Has anybody ever encountered this situation before? Do you think if retroviral DNA get methylated by the cells when they are incorporated into the genomic DNA?
Also, any things to look out for when using retroviral vector to make stable cell line?
Thank you very much
Firstly, check that the retrovirus that carries you transgene is expressing the proper protein.
Do a quick RT-PCR to check if the transgene is intact, some retroviral vector are known to splice the gene you inserted into it.
To confirm, do a western of the packaging cells to look for the protein, run it alongside a positive control of the protein. If all is good, then there are other possibilities that your protein is not overexpressed in the target cells.
Try to sort the cells for a highly expressing population (by flow cytometry) if there is a suitable fluorescence marker.
Otherwise, do a serial dilution to get almost one cell per well in a 96 well plate and then culture each clone into a larger population , enough to run a dot blot, then keep a few high expressing colonies for you future works.
Hope it helps.
Hi Anh,
In addition to suggestions above, is there a tag in retroviral vector, such as GFP or RFP? If yes, can you see the signals under microscope or by FACS? Another question is, what's the basal level of the protein in cells? It is quite difficult to see big differences if endogenous protein is highly expressed in control cells. Lastly, overexposure of the films when doing WB also makes no difference between groups.
Unfortunately my vector does not have any fluorescent marker. It does have an antibiotic reporter gene for positive selection and that's about it.
the protein being expressed is a signalling molecule so Im not sure if the cells somehow adapt to this and degrade the proteins and bring back to the normal level?
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