Hi,
I´m searching for a vector that can be used to express two proteins via different promoters in mammalian cells. My goal is to generate a stable cell line. I already tried an IRES, but I only got a very weak expression.
Hi Stephan,
An IRES is designed to load ribosomes on already transcribed transcripts. I have had problems getting expression of 2 genes using 2 CMV promoters. This has been referred to as squelching.
To get around this I have used a CMV promoter to drive one gene and a HSV-TK promoter to drive another gene. You could then insert and IRES to enhance protein expression of the message. I think there are some lentivirus backbones with 2 promoters built in, but they end up being very large constructs.
Alternatively you could use a T2A system to cleave the translated proteins and get two separate protein products from the same message.
Hope this gives you some ideas.
Thanks man, I tried a P2A site and it works just fine!
Best
Stephan
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