Dear Worm Community,
I’m interested in swapping an endogenous 3’UTR with a highly expressed 3’UTR (e.g., tbb-2) to enhance gene expression. I was wondering if anyone has experience or insights on whether inserting a 3’UTR between the stop codon and the target gene’s native 3’UTR would work, or if it might result in an mRNA with two 3’UTRs, causing expression to follow the native 3’UTR.
Any suggestions, comments, or similar examples would be greatly appreciated!
Hi Ken,
I’ve done it with a few germline-expressed genes. In my case, it was ineffective at dramatically increasing protein expression but your mileage may vary. As long as you include the poly-A signal of your integrated 3’ UTR, (should look something like AAUAAA) the endogenous 3’ UTR should be cleaved off of the messenger RNA. However, I did not directly confirm this in my case.
Ian
I just looked at it and tbb-2 3’ UTR does not have a canonical poly-A site. Regardless, integrating the 3’ UTR should generate a message without the endogenous 3’ UTR.
If you are curious, this paper has good information about poly-A sites and 3’ UTRs in C. elegans
The C. elegans 3′ UTRome v2 resource for studying mRNA cleavage and polyadenylation, 3′-UTR biology, and miRNA targeting
Hi Ian,
Thank you so much for sharing your experience and for providing such helpful information.
Your explanation helps clarify what actually happens and how I should design my experiments in practice.
I also really appreciate your effort in checking the tbb-2 3’UTR and for sharing the reference—it’s very useful.
My recent observations also suggest that swapping the endogenous 3’UTR doesn’t always lead to a dramatic increase in expression in germline-expressed genes, which aligns with your findings.
Based on your insights, I may explore alternative approaches for achieving overexpression.
It’s reassuring to know that my results are in line with what you’ve found, and your feedback has definitely given me new ideas for next steps.
I truly appreciate your time and valuable input.
Thanks again!
Ken