Transient Transfection into HEK -> EL expression - (Aug/13/2008 )
We have an identified plasmid (vector pcDNA 3.1/myc) that's carrying a gene to express Endothelial Lipase (EL). We're using Lipofectamine 2000 to transfect the plasmid into HEK-293 cells, allowing 24hr growth in serum, then incubating them in a serum-free heparin solution so that the cells release the membrane bound enzyme EL.
However, activity assays using the collected media only result in background. We're using enzyme produced in the same fashion by another lab as a positive control, so we know that our activity assay is on target. I was thinking that if we introduce ampicillin during the growth phase, after the HEK cells had been transfected, we would be able to select out cells that weren't accepting the plasmid to see if our lipofectamine is working properly (it is somewhat old, but expensive) - but I don't know if HEK cells would grow in the presence of amp, with or without the plasmid.
Any ideas or suggestions? Thanks in advance,
Have you checked the expression of EL in the cell lysate AND in the medium? I mean how did you make sure it's a secretion expression, or it is even expressed?
Since you are using pcDNA 3.1, why don't you use G418 selection to establish a stable cell line if you want to?
Ampicillin is for the selection of recombinants from transformed bacteria, it's just a kind of antibiotics. I don't think it can kill the mammalian cells, otherwise when we take ampicillin pills......
Do a transfection control with GFP? Did you check expression in cells after transfection before adding the heparin solution?
After transfection, i would leave the cells for upto 36-48 hrs for maximal expression.