I wish to make stable Huh7 or HepG2 cells with my gene of interest using pcDNA3.1(+).
I´m using Lipofectamine 3000 kit and Geneticin selection 72h post transfection but the cells tend to loose the gene after few passages.
Anyone has experience in stable cell line generation with this kit and can suggest me the best performing method to have a stable cell line?
Thank you in advance
For a stable cell line, we use lipofectamine to package virus components into 293T for virus packaging, virus particle will be generated and released into supernatant. The virus will then be used for stable cell line production (polybrene; spinfection, etc). Please take a look into pseudovirus production protocol.
Hello Ilaria Rossi
You cannot prepare a stable line by simply transfecting pcDNA3.1 using Lipofectamine. This will definitely transfect your cells, but it'll be an unstable or transient one and your cells will ultimately lose the plasmid. You need to generate retroviruses that will help in integrating your gene into the host genome. This way your gene will be integrated and stably expressed.
You can certainly make stable cell lines with plasmid integration.
However, it generally takes about 2 weeks or so with constant selection, and yes, a vast majority of cells will die/lose expression. That is completely normal, and a low percentage (0.1 to 2%) will integrate and become stable. You can also linearize your plasmid before transfection and this does increase the overall efficiency of generating stable cell lines.
The advantage is that you often get multiple copies integrated, and therefore higher expression, and can be a faster alternative if you do not have any viral transduction system in place. The disadvantages: Colony founder affects, variable expression, and it takes 2-3 weeks.
In your case, 72 hours is no where near enough time, and you should get some colonies growing in about 10 days or so.
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