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Express two different membrane proteins on the same promoter


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#1 anonyvous

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Posted 25 July 2017 - 05:45 PM

How could I generate a stable mammalian cell line that expresses two different membrane proteins on the same promoter? I want to know what strategies I could take.



#2 bob1

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Posted 28 July 2017 - 11:38 AM

There are a few you could take, it depends on what sort of line you want to take - do you care how many copies of the genes are inserted? Where they might be inserted in the genome? Do you want an inducible/repressible system (switch on when wanted or off when wanted)? Do you want these genes expressed off one transcript or two (two plasmids running the same promoter or one plasmid expressing both genes?)?

 

Most systems will insert multiple copies of the genes into random places in the genome, but things like CRISPR/CAS and Cre/Loxp can be used to insert single copies. CRISPR results in site specific insertions, so you can choose where they go in, which can be important if you want high level expression - not all genomic sites are equal in this respect. Standard transfection and viral systems that use antibiotics to select for the transgenic cells usually result in multiple copies being inserted, insertion in these cases is random and usually heterogeneous in terms of expression levels in any specific cell within a population (i.e. one might be high, the next low). Insertions that are based on bacterial and viral systems are often preferentially methylated and suppressed by the genome too, so you might want to consider if this is likely to be a problem or not.

 

Inducible systems have problems too - some are leaky (e.g. tet repressors/inducers), some are not so leaky.

 

With  a single promoter you usually need something like an IRES sequence between the genes so that you get independent expression of the transcripts - though this often results in the second being much weaker expression than the first, there are other options for this which seem to be less affected in terms of expression levels, but I'm not really familiar with them.






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