HEK293 transfection using electroporation. Science or magic! - (Aug/09/2007 )
I am using HEK293 cells maintained in Freestyle 293/5%FBS medium. Cells are harvested at 80-95% confluence and adjusted to 1E6cells/0.6ml freestyle 293 medium (without FBS). These cells are distributed in 1.5ml sterile eppendorf tubes on ice and added with 3ug plasmid DNA. My plasmid is ~6.7kb and has an HIV-1 gene fused with GFP under the control of CMV promoter. Cells/DNA is transferred to prechilled electroporation cuvette (0.4cm) and electroporated (400V/975 uFD. ~30ms). The cuvettes are immediately transferred to ice and allowed to stay for 5 minutes. After that I transfer the cell suspension (avoid foam/froath on the surface) to 1.5ml eppendorf tubes and spin at 3000 rpm/5 min. The sup is discardedand cells are resuspended in 1 ml of pre-warm Freestyle293/5% FBS. The suspension is put on collagen coated coverslips and incubated at 37oC/5%CO2 for 48 h.
The expectation is to see GFP both inside the cell as well as in the spent medium.
Transfection effeciency has been fluctuating between 10-70% (with same DNA prep)
Even thouhg cells are transfected (green under Fluo microscope), no GFP secreion is observed (using fluorimeter).
What is the fuss?
Another fellow in the lab has originally developed or established this protocol. Unfortunately, only he or his God knows, what exactly he does since he always gets very high transfection as well as secretion of GFP fusion protein in the sup. Problem is, he never shares the exact protocol and keeps modifying it. Initially cells were maintained in FBS free media (even then she was getting results), then he started maintaining in 5% FBS. Initially he used 1ug DNA/1E6 cells, later he said 2-3 ug DNA. He never counts the cells rather "feels" them. In the lab meetings, there are numerous theories. Maybe cells are old (high passage number), maybe cells are harvested at wrong time, maybe cells were overexposed to trypsin, maybe DNA quality was poor. I have tested each of these one after other but boy, I could'nt solve the mystry. I am really frustrated at this point. It has been almost a year of unsuccessful attempts on this issue. Anyway, I'll highly appericiate if somebody could help me out.
Are you 100% sure about the right localisation signals in your protein of intrest? Not all HIV-proteins will be automatically secreted. (the purpose of the proteins is to be inside the virion; to regulate gene-expression from HIV-genome once integrated or to down-regulated CD4 and MHC's, though some will leak into the medium)
Have you considered trying a different method for transfecting your cells? 293's are easily transfected with high efficiencies using cheap methods (Calcium-phosphate with home-made solutions is the one I do regularly, but probably other methods will work too) as well as with reagents (Lipofectamine, fugene and the likes).
Does your colleague also do this experiments with the exact same vector?
Have you done blotting with your medium to find if any protein is around? (maybe your fluorimeter is not sensitive enough)?
OK, do the following:
1. have your colleague do the experiment with your reagents and her (cells, solutions, everything)
2. at the same time you do it, with her reagents and yours
3. follow what she is doing exactly (write it down)
That should tell you if there's a problem with your reagents. If there is no problem with reagents, then it must be your technique, but now you should at least have a reasonable idea of a protocol.
It's quite common for beginners to have less transfection efficiency than more experienced people, however the senior person in the lab should really be teaching you how to do it, not let you stumble around in the dark. Sounds from your description as if she is unwilling to help you. Did you do anything in particular to piss her off? Maybe infer that you knew better than a woman anyway or better than a Biologist if you're a Medic? Or maybe she's a research assistant and you've been lording it over her?
Because all of that has happened to me a few times and my usual reaction is to not be actively helpful to this student and just let them run into problems on their own. At some point they'll realise that more experience in the lab isn't gender dependent or that Medics aren't automatically right or that pissing of the RA is a really bad idea and come back asking for help...
Maybe it would be a good idea to sit down with her in private and discuss your working relationship? If that doesn't help sit down with your boss or even the graduate tutor (if you are a graduate student) or some other person designated to be a counsellor. If you've spent a year on this already you obviously aren't getting the supervision you should have (sadly that happens quite often).
In an aside your colleague really should not change the transfection conditions all the time - how are experiments comparable if she does?
Hope that helps