Hello everybody,
I am trying to express several recombinant proteins in HEK cell line by transient gene expression. I am using as medium FreeStyle 293F to produce the proteins by polyethylenimine (PEI)-mediated transient gene expression. From some time now, the expression is not working. I had successfyll expressed these proteins in many occasions before; using the same medium. Currently other collegues who are working in the same lab do not have any problem (working with other medium). Did anybody have a similar problem?
Thanks in advance!
Best wishes,
KIka
You have to troubleshoot your problem step-by-step
- quality of your DNA prep
- problems with the cell line
- media composition
- detection
If you have colleagues in the same labs who have no problem, you can try to exchange individual components to see which one is related to the problem
It is hard to diagnose other people's experiment. HEK293 (Freestyle 293F?) should be easy to transfect and the heterologous protein expression from CMV or EF-1α promoter driven gene is usually very high. Among different HEK293 derivative and transfection protocols, we found the Expi293F cells using Expifectamine to transfect gives us the highest yield and more consistent result. For large scale production, we would use electroporation instead to lower the cost.
You can try isolating fresh plasmid for transfection so basically, it is your quality of DNA as also noted by Annemarie Honegger
You should specify some details - did you prepare a new PEI stock? Does transfection succeed with other plasmids (e.g. GFP)?
Did you test HEK293F for mycoplasma contamination?
Did you use a kit for low-endotoxin DNA purification?
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