Hi,
I have transfected HeLa cells with pCDNA3 eGFP by a calcium phosphate transfection protocol. I was wondering if I can maintain the fluorescence expression over time by passaging. Every week, I transfect a new batch of HeLa cells with the plasmid and this takes considerable time and labor on my side, so Im thinking if passaging these transfected cells would be okay to do to save some time and effort. I've never tried it so I'm happy to hear your opinions on how to go about this.
Thank you very much!
There is a distinction between consistent transient transfection from repeated transfection of cells and stable expression of a gene of interest. For HeLa, where transfection efficiency should be over 90% (https://altogen.com/product/hela-transfection-reagent-cervix-adenocarcinoma-cells/), the transient effects will be very apparent. However, maintaining cells with expression will require subsequent selection to make sure the cells that remain are stably expressing the gene of interest.
The plasmid should allow the expression of eGFP as long as the plasmid is intact/present in the cell, and eGFP has a relatively long half life, often more than 24h. What is the purpose of your transfection? Are you also trying to express a particular gene with the eGFP label? Depending on your answer, I would probably err on the side of caution and transfect for each experiment in order to ensure quality, trustworthy data.
Dear Alvarez,
In my experiments with HeLa transfected with pCDNA3 beta-eGFP plasmid, the GFP expression remains till 72 hours after transfection. Normally I observe the highest expression in between 36-48 hours, after which it starts declining. I think till 3-4 days you can still expect some GFP positive cells. If your experiments demand and if you think it will make things easier, you may establish stable cell lines using G418, as pCDNA3 beta vector carries the neomycin resistance cassette. But you need to decide depending on the experiments you want to do with them. Hope it helps!
Dear Kris,
it really depends on what you are trying to do in your experiment, but it does sound like making a stably transfected cell line as Tias already suggested would be the way to go.
Do do this, you should linearize your plasmid (not in the neoR or your promoter&gene of interest), then transfect as normal, wait 48 h and then apply G418.
I've attached a intro guide on how to make stable cell lines, but there's loads of info available on the web if you look for generation of stable cell lines.
You can try to use lentivirus system if you want to gain stable eGFP in your experiment Hela.
I really don't understand why you would want to make a lentivirus to get a stable cell line in easy to transfect cells like HeLa? That's loads of extra work/expense for no gain...
If you are trying to transfect difficult/primary cells, then yes, lentivirus might be the way to go
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