I have pcDNA3.1+ Insert + GFP vector. I transfected this vector into A549 cell line. I got minimum GFP expression after 48 hours (only 5 to 10 cells give GFP fluorescence) and I trypsinized that cells to transfer into petri dish with G418. I maintained the cells one week (3 day- media changed). On the 7th day the cells detached from the dish. Any idea why?
I used lipofectamine 2000 invitrogen for my transfection.
1). Can anyone give me suggestions for stable expression cell line maintenance, briefly?
2). Which is the best best eukaryotic expression vector?
3). How to increase transfection efficiency in my vector construct (pcDNA3.1+insert+ GFP)?
Hi senthil,
Since you placed your cells in a selective medium containing antibiotic, all the cells which are not transfected die as they donot have antibiotic resistance for G418. if you think your GFP expressing cells are dying, then you have to adjust the dose of g418 for the cell line you are dealing with. I use Fungene HD wich has a pretty good transfection efficiency.
I am not sure I understand... Are you using a single vector, or is this a double transfection protocol? By this I mean: are the GFP and the insert cloned into pCDNA3.1? Or are you transfecting with more than one vector?
Plasmid integration into the host genome is a rare event. You'll need tens of thousands of positively transfected cells to be able to generate stable cell lines. Try using linearized vector instead of circular DNA. Make sure you've optimized your transfection protocol (you may need to try electroporation or nucleofection instead). As Shiva suggested, using the minimum amount of G418 necessary for selection (make a killing curve with non-transfected cells first).
If you cannot achieve high transfection rates with plasmid DNA, you may need to switch to a viral vector instead. Most viral vectors can achieve high rates of transduction and are stable for weeks (adenovirus or AAV) or longer (retro and lentiviruses).
In my experience, the process of selection takes much longer, because untransfected cell are not killed at once. Give them at least 2-3 weeks and keep them in selection medium. The right concentration is also important.
As Daniel suggested linearize your vector first, which prevents random linearization which might destroy your selection marker.
I can recommend Lipofecatmine LTX (tested on MDA-MB-231 cells).
Guillermo Romero. yes my gene of interest and GFP was cloned into pcDNA 3.1+
i was transfected only one vector pcDNA3.1+ vector..
Senthil,
We do exactly what you said you are doing all the time, but we transfect with Fugene, and use different cell lines. After selection with G418 for 2 weeks+, we select clones because gene expression is not the same in all cells. Usually we just select 2 dozen or so clones and look at them with a fluorescent microscope, keeping the brightest ones. If your gene is fused to GFP, that is enough. If not, you need to check expression of your gene of interest by Western blots or qPCR. In general, close to 50% of the clones we select are resistant but do not express substantial green fluorescence. We don't bother with retroviruses or lentivirus unless the cells are primary, or extremely difficult to transfect (like MEFs, or some smooth muscle cell lines) or linearization protocols.
Caveat: we have no experience with A549 cells. Good luck.
I forgot to add: wait 48h after transfection before you start adding G418.
What Guillermo said. Depending on your project you may however also want to keep some clones with low expression since whatever construct/insert you are expressing may otherwise be excessively abundant when compared to the cell-intrinsic native protein corresponding to your insert (if that is applicable to your situation).
In addition, a quick Western blot on the clones is helpful as well to ensure that you GFP-fusion protein is expressed at the correct size. I had clones myself where only truncated variants were expressed.
A549 is quite commonly used and to my knowledge can be transfected fairly well. We only ever did it transiently in these cells, though.
Dear Senthil,
for plasmid transfection of A549 cells I advise Viromer RED (from Lipocalyx).
Viromers are syntehtic polymers with an active endosome escape mechnism. By using Viromer RED I got higher transfection efficiency compared to standard reagents.
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