I need to transfect (Lipofectamine 2000) 4 expression plasmids (totaling 10.1 microG all containing CMV or CMV minimal promoters) into 293T cells (>90% confluent at time of transfection in 10cm dishes). Expression vector A has 8microG delivered; expression vector B has 100ng delivered; expression vector C/D has 2microG delivered. When I transfect only vector A and B I see normal expression of all proteins (total 8.1microG). When I transfect either vector C or D in addition to A and B (total 10.1microG) I lose expression of vector B. Vector C contains a CMV promoter but lacks a gene cassette. My question is this: what is the maximum ability of 293T to express a CMV promoter? Am I saturating their ability to express my proteins? What other possibilities exist that explain this phenomenon? How can I successfully express all 4 proteins in my system?
Did you already try to leave a day in between the transfections? Meaning: Transfect vecor A and B on the first day, transfect cells with vector C or D the next. Worked for me once.
however, if I understood you right, you transfected your cells with only 100ng of vector B but 8µg of vecor A and 2µg of vector C or D?! this is a 20-80fold difference. overloading cells with plasmids is never a good idea. so maybe you should try to decrease the amounts of A, C and D and perhaps increase the amount of vector B to maybe 500ng to 1µg!
how large are the individual plasmids? if they all have approximately the same size and all have a CMV promoter try to transfect the same amounts of plasmid. Good luck
We are using BacMam method, a single virus contains all gene cassettes. And it is much cheaper way if you need express the multiple protein often.
Hi Kristina,
For a T75 flask we use 40µg plasmid and PEI MAXX (1:2.5 but you may need to test it with your cells and media). We use the plasmid with our gene of interest (90%) supplemented with 10% untagged GFP plasmid added as a transfection control. We normally see >80% GFP positive cells so even with 4 plasmids I think you would still obtain a reasonably high percentage co-transfected.
Not really sure of the rationale behind changing the ratio of the different plasmids. You may get different expression levels across the whole pool as different cells may be transfected with different combinations but if you want to investigate the effect of expression levels of one on the levels of another this is probably not the way to go.
To make it more robust you could try cloning the 4 genes into only 2 plasmids with IRES/IVS between each pair of genes.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression