Hello everybody,
I am currently doing interaction studies of two proteins which I am overexpressing in HEK293 cells. So far, I have been co-transfecting my cells with two seperate constructs. To ensure that all transfected cells express both proteins, I was thinking of cloning both cDNAs into the same vector instead. Does anyone have any experience with this? In my mind, I don't see why it wouldn't work to just put two expression cassettes in the same plasmid, assuming the final construct still has a reasonable size.
My main question would be about how to design such a plasmid. Should I try to put both cDNAs in tandem between the CMV promotor and the poly-A signal/terminator? Or should I rather put in two individual cassettes in, each having it's own CMV promotor and terminator?
Any suggestions or supporting literature would be greatly appreciated, thank you!
Putting both cDNAs in tandem but separated by a T2A, E2A or any 2A based skip peptide is a good idea. But if the size of an individual gene exceeds 2-3 kb, the protein expression may not be very good.
In such cases both genes can be cloned into separate cassettes. This is done for dual heterologous protein expression in bacteria. Have a look at the pET Duet dual protein expression construct for bacterial systems.
https://www.snapgene.com/resources/plasmid-files/?set=pet_and_duet_vectors_(novagen)&plasmid=pETDuet-1
Totally doable. You need to use any multicistronic vector and clone your genes accordingly (https://blog.addgene.org/plasmids-101-multicistronic-vectors). These vectors have IRES inserted between two genes.
You can try what has been suggest above 2A skip peptides or IRES, however, with 2A peptides you will add some amino acids to your proteins and with IRES you will get lower expression of one of your genes dependent on its position relative to the IRES. You can try a bidirectional CMV promoter like in plasmid pBI-CMV1 (see link for map) maybe this will do the trick for you.
https://www.snapgene.com/resources/plasmid-files/?set=mammalian_expression_vectors&plasmid=pBI-CMV1
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