I have created stable cell clones of a protein whose gene was cloned in pcDNA3 which has G418 resistance. I started observing colonies after 15-20 days of growing them in the medium. The cells inherently also express the protein and I am trying to over express that protein. Now when I have started for screening of the stable clones which should over express the protein, I see a similar pattern of expression as that of the control. Can anybody suggest why the protein is not being over expressed despite resistance for G418?
when you establish stable cell lines, In order to achieve homogenous over expression. As an initial screening you should pick up multiple colonies and grow them individually (because each colonies may have different expression levels of proteins). Then compare the expression levels by Western blots and immunoflourescence microscopy. Scrutinize and then carry out your experiment using the cells with high expression level.
@ru-jeng teng: is it possible to screen transiently transfected cells by G418?
You should check the expression of several clones by western blot in order to confirm an increase in the expression. Do you have some tag in your pcDNA3 vector? Some pcDNA3 carries Cmyc, Flag, etc, so, you could check specifically your overexpressed protein by using specific anti-tag antibodies.
Hi Mansoor: I would like to know the answer myself. Our group havbe tried that a few times but found out that the protein production disppear after a few days. That made us puzzle whether what we had was just a transient transfection.
Try varying the amount of G418 used for selection. 15-20 days in culture seems like a long time to get colonies. My guess is the selective pressure is not strong enough. Perhaps your transfections are not working properly. Westerns seem like the best bet, but RT-PCR for your gene of interest may be an option.
Stable mammalian cell lines are difficult to obtain by using the conventional expression vectors such as pcDNA3/pCR3 and derivative versions. Lucky enough after screening for ~hundred clones, you may get a few positively expressing your protein/gene of interest. This is probably due to the strong CMV promoter, the cells simply don’t like your protein being highly expressed in long term. What is likely happening is, under the selective G418 pressure, the cells can survival because of expressing the resistant gene but not your gene/protein. As suggested by others, transient expression should be performed.
There are still some precaution;
1. Make sure the antibody is good enough for WB, include positive control sample(s), Eg. cell lines previously found to express your protein, or your purified protein (see if commercially available or available in other lab). Or you tag your protein as suggested.
2. Perform RT-PCR as suggested, make sure the gene is actually expressed
Good Luck!
I have done the western blot and on that basis only i am saying that the cells are not over expressing the protein. also, the cells inherently express my protein but ectopic over expression is not being observed. the issue is that
So your recipient cells constituently express your protein and you want more protein to be expressed by using the pcDNA3 vector. Apparently, from what you said the antibody/WB works fine. As I said, in long term your cells cannot tolerate the higher levels of expression. As a result, the drug selection process has selected the fittest cells to survive which are the cells without overexpression. For transient expression, the high protein levels should be able to sustain for a few days and that is why some of us suggest you to do so. What is/are your subsequent assays? If you really need stable expression you can try the lentiviral expression system, which is a robust method for overexpression in mammalian cells.
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