We are working on muscle nAchR and have plasmids for 4 subunits i.e. alpha subtype 1, beta subtype 1, epsilon and delta. In addition to this we have a CFP plasmid. Untill now we were doing transient transfection in HEK-293, but now we want to have a stable cell line. Is it possible to have stable cell line with all five?
If it works transiently, it should be possible stably as well. But how much work this is gonna be for you depends very much on the system you want to reconstitute (i.e. how important the correct expression level is for your components). basically, you should generate lentiviruses expressing all 4 of your constructs and then generate a monoclonal line with the first component and test it by cotransfection with the other 3 constructs. Then take a good clone, transduce it with the second construct, find the the best clone by transfection of the other 2 constructs and so on. this might well be a year of work, so in the first place you should think whether it's worth it. If the individual expression level is not that important, you can use a combined construct as a shortcut (i.e. one construct with several of the subunits linked by p2a-peptide sequences or 2 subunits linked by an IRES - in the latter case, the second protein will have a lower expression level thant the first one).
If you prefer stably integrated plasmids over lentivectors, you will in my experience need approximately three times longer and have to screen many more clones (though it might be an advantage if you go for the all-in-one construct, as lentiviruses can hardly handle cDNA inserts >4kb).
Good luck with your project. Best, Thomas
Just one question here, for such hetro oligomer, i suppose your protein is pentamer how you control the stoichiometrie and correct organisation of your each subunit in pentamer irrespective of transient or stable cell line.
I know of multibac Expression system ( Imre Berger Group) used in multiprotein complex as a solution to stochiometry problem of hetro and multioligomer, this has been used widely in soluble protein and to some extent in membrane protein, one should give a try. Ligand binding at the end will be definitely used to check functional receptor.
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