Need a weak promoter for reporter gene assay - (Apr/16/2007 )

Dear friends,

I need to study a molecule's effect on promoter activity, most likely the effect of activating the promer, using reporter gene assay. If I use a strong promoter probably it is hard to detect any changes because the reporter gene activity is already very high. I wonder whether there are "weak" promoter sequence which just has some basal activity so that I can insert it into the reporter vector.

Thanks in advance.

If you are studying effect on promoter activity, maybe SEAP assay (Clontech) could be of help since it is easy, don't have to lyse cell, and quite sensitive. The SEAP construct is with SV40 promoter as well. I think that it had been published, and at least in our case we have seen the difference, that SV40 is a weaker promoter compared to CMV. If you use SEAP and analysed in either a short enough time or enough dilution, it should be sensitve enough for your need. The problem is that you can't make stable cell line with the SEAP construct that is provided, so may need to use luciferase for transfection control or have to put the entire encoding gene, promoter, enhancer... into your choice of vector.

Thank you both for pointing me to the SV40 promoter. I will give it a try by insert it into a luciferase or GFP reporter vector. Probably I have to add some sequence upstream the SV40 promoter which should be very short. The added sequence will contain the binding sites of my interest.