Gene promoter - Luciferase reporter assay help - (Jul/10/2018 )
Hi there, I've never done a Luciferase reporter assay before and am also new to DNA cloning etc, which is why I'm very confused.
I have a plasmid with my gene promoter of interest linked to Luciferase reporter in the pGL3 basic vector, available from Addgene. I need to express it in HEK 293T cells, which are human cells. However, on looking into it further, it turns out that the only gene promoter available on Addgene is the mouse version.
I wanted to see whether my stimulus will induce transcriptional activity in my promoter (demonstrated by luciferase activity). However, having seen this is the mouse version of my promoter gene, I am wondering whether this experiment will work in human cells?
Could someone please shed some light on this? Any additional info on the assay protocol would also be useful.
Thanks so much
It probably depends on your promoter, specifically, as to how well it would work in another mammalian species. I suggest you look at the references for the plasmid- from the lab constructed it. Read any publications to look for clues on how species-specific it is. Did they use it in mouse cells or others? If that doesn’t help, see if you can contact any of the people who deposited it for their opinion. Another Source of information: contact Addgene itself. They have a PLASMIDS 101 educational resource and forum: https://blog.addgene.org/plasmids-101-the-promoter-region
You may find that it would be best to match the promoter species origin to the cellular origin. If you must use the human cells, and if you can get the nucleotide sequence of the human promoter from GenBank, maybe you can design PCR primers and amplify the human promoter out of some genomic DNA derived from those cells. You could add convenient restriction sites in the primers and clone it into the position now occupied by the mouse promoter in the plasmid. You’d have to sequence the new fragment to confirm its sequence.
Thanks for your suggestions Old Cloner. Problem is I've never done any cloning before and have very limited time to get the results for this experiment. Do you have experience in carrying out luciferase assays?
Not really- I have built a lot of expression and reporter constructs for the use of others (that was my job) but the downstream stuff in eukaryotic cell culture was in the client's hands. Hopefully someone with that experience will jump in here!