Hi everyone, I am a newbie in molecular biology. Recently, I encounter an issue that the non-uniform fluorescence protein expression level in lipofection transfected HeLa cell. The variation would cause serious problem during the image processing. I plan to make a monoclonal stable cell line which should have a more consistent expression level.
I would like to know if there are any plasmid design rules that I could follow or reference? I know that viral method is one of the popular method, however, I prefer to use non-viral method. It would be good if I could just do lipofection and perform selection afterwards. Then, generate the monoclonal cell line by seeding single cell in 96 well plate.
In theory, your approach should work with any expression plasmid. As you described, you need a selection marker. Some plasmids might have a higher chance of integrating into the cells genome, but it should work with your regular expression plasmid. I would suggest that you simply try with the plasmid you have. It is really a game of chance, so transfect a larger batch of cells. Then select for a couple of weeks before isolating single cell clones.
Remember though that the plasmid DNA will integrate into the genome randomly and can thus damage endogenous genes. This is no issue if you just want to express protein. However, if you plan of using the cell line for other experiments, I would suggest a more elegant and targeted approach to rule out any possible side effects.
Good Luck!
Dear Dr. Chow,
I attached some of my protocols on this subject.
Best Wishes
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