I never tired this.

But take into account IRES leads to low expression levels and two IRES might lead to even lower ones.

Have you considered using a larger vector with two promoters instead? If you add IRES under both promoters you can even express four proteins.

You might also want to prefer co-transfection of several vectors.

Unless, you really just want to know if it's possible or not, which is also cool :)

Good luck,

Guy

Hi David,

Like Guy, I have not tried this either but what you should know about IRES sequences is that the proteins after the IRES sequences will not be expressed at the same levels as the protein directly after the CMV promoter. So if you need all three proteins expressed at the same levels then using IRES sequences are not the way to go. If you need equal levels you should consider putting viral 2A sequences between your open reading frames. I know that 6 proteins have been expressed at the same time with 5 viral 2A sequences between them and at equivalent levels.

Good Luck!

Hi. I know, I tried one IRES and expression was very low. But there are a lot of IRES and maybe that was not a good one. Now I tried another IRES using mCherry and expression was decent.

So far, as you mention it, I used two independent SV40 promoters for the two independent proteins but you know, how I can know for sure and quick that both are expressed? the second protein is EGFP tagged and the first is not. I see faint green in my cells and I am left scratching my head.... Now I have to check the first one by western but its tedious...Im afraid that maybe one protein is expressed high and the other not...

I wanted to devise a system that would ensure that if I see fluorescence in the second protein means the first is also expressed...

thanks for your help Guy

d.

Hi Tom.  I have been reading about the 2A peptides and is something I am considering doing, who managed to express 6 proteins using it? Can you send me the pubmed link?

Thank you

d.

Hi David,

Attached is the paper I am referring to. It's really a great way to express multiple proteins from the same promoter. I made a CMV driven mCherry-2A-GFP construct and both fluorescent proteins were expressed at the same levels. Worked like a charm.

Good Luck!

Thank you Tom. I really appreciate your help. Would it be possible to know the sequence of your construct and In which cell did you expressed it?

Thank you

d.

Hi David,

Here is the sequence of my mCherry-2A-GFP construct. I have expressed it in both CHO and HEK293 cells and it works great in both.

Let me know if you have any other questions.

Tom