I am trying to co-transfect 2 plasmids in HEK-293 cells. One is pcDNA-3 based( protein A) and the other is pcDNA5/FRT/TO based GFP-tagged ( protein B). Protein A's expression is optimized. Protein B's expression is yet to be optimized, but the plasmid works. What are the basic rules i have to keep in mind while doing this co-transfection to obtain equivalent yields of both proteins. I have to study protein-protein interaction after subjecting the cells to a certain treatment. Also FRT/TO based vectors are TET inducible. So when and in what conc. do i add Tetracycline?
Hi Bhaskar, I cotrasfeccted hek293 and CHO cells with two plasmids encoding heavy and light chains of an antibody, the main factor affecting is the relation (in amount) among the vectors and must be optimized because the expression is affected by many factors: promotor of each protein (I think the promoter are the same for your bouth proteins, so this dont apply for your case), size of the protein (In my case, small proteins exprres in higer quantity than largest ones), the cell line, concentration and purity of the plasmids, and the transfeccion method.
I recomend this papers:
Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability.
On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells.
Best regards
Thank you for your answer. I actually need to know more about the pcDNA5/FRT/TO system. is protein B tetracycline inducible in HEK cells AS WELL?
Hi Bhaskar,
Yes, protein B in the vector pcDNA5/FRT/TO can be transfected, expressed and also induced in HEK-293 cells using TET.
- Badges
- Science method