Dear all, I am working on making a construct with sra-6 promoter, to drive my gene of interest to express in C. elegans ASH neurons. When deciding the length of the sra-6 promoter, I searched the web. Lots and lots of paper did not indicate the length they used, while in the a few papers that did mention they used either 3.8 kbp or 4 kbp.
4 kbp is safe in general, but when I checked it on Wormbase, the 4 kbp upstream of sra-6 contains two other complete genes (F35H8.12 and F35H8.1, see attached figure).
Since lots of people might have experience with sra-6p, I figured it is faster to ask here, that:
What is the sra-6 promoter length did you use in your lab?
If you used 4 kbp, does it matter if it contains other genes in your promoter?
Has anyone used a “cleaner” version of the promoter, maybe less than 2 kb, and did it work?
I can’t answer any specifics about sra-6, since I haven’t worked with it. What I can say is that the upstream gene that’s in the opposite direction may share a promoter or enhancer with sra-6, or they could be two independent units. I would say that looking at the modEncode data, to see where known transcription factors that affect sra-6 expression (if any are known) could give you a better indication of where the regulatory elements for sra-6 are, and if a 2kb version of the promoter would be sufficient.
That being said - if what matters to you is the expression pattern of the sra-6 promoter in the literature, rather than the true regulation of sra-6, then I’d just use it as is. If you go less than 4kb there could be elements lost that are important for making the expression pattern as published.
That said, if you do use the full 4 kb it might be a good idea to insert stop codons or frameshifts into the other open reading frames so you you don’t have to worry you’re doing anything to the worm by overexpressing those proteins.