Hello, in my expression cassette I already have 2 proteins that are expressed using IRES. However I'd like to express another protein at the same time (a homing molecule), but, honestly, I do not know if it's possible. Since this is for clinical purposes use I cannot use co-transduction, which is the major hurdle since I am limited to using only 1 vector. Is there a way to overcome that problem? Thank you.
Hi Mark,
As I see it you can look into these possible options:
1. add another IRES+Gene after your second gene. This should give you expression of all the genes, however the level of expression will be low, and might not give the required results.
2. add a second promoter+GOI to the vector to express your gene. This would give you more control of the expression but there is a chance of silencing of the expression.
3. if you already have another expression cassette (e.g. for selection) add the IRES-GOI to that. Might not be an option as you say that it's for clinical purpose.
(Note: I've never constructed a vector for clinical use, so don't know if there are reason for not doing any of these in such a setting)
-Richard
Hello Mark, there might be conditions preventing you from doing this, but have you thought of using two promoters? One for the bi-cistronic cassette and another one for the third ORF. The maximum payload your vector can carry and parallel promoter orientation to avoid plasmid/vector instability are points to consider.
Richard and Balazs have good suggestions indeed. Alternatively, you could use 2A peptide between 2nd and third cistron.
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