I am trying to express GFP, Luciferase and Puromycin resistance from the same plasmid using sequentially 2 T2A peptides in between. Has anybody experience in such a construct design and have any suggestions/comments?
This should be possible. Here is an example I found on Addgene that incorporates mCherry, Puro, and RLuc into a single transcript (https://www.addgene.org/29783/).
One thing to note is that 2A sequences also cause ribosomes to fall off during translation, leading to lower expression of the downstream protein. Adding two of these sites to a single transcript will compound this effect for the third protein. I'd recommend putting Puro as the second protein in your proposed plasmid to ensure you get a high enough expression for antibiotic selection.
Do you have to use T2A peptide linker? Are you planning to make a stable with such construct? For promoter trap? Otherwise, you can use an IRES vector for expressing the 3 proteins from the same plasmid under 2 promoter control. The second generation of IRES-EGFP, which is from around 20 years ago, has improved the translation efficiency of the second gene in the same transcript.
HI, not sure if you still need it, but I was encountering a similar issue.
Turns out, it is possible, but the efficiency of ribosome skipping decreases the further C-terminal you go. I'm linking below a paper I found useful to decide how to organize your 2A tags. Hope this helps.
Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector | Scientific Reports (nature.com)
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