Luciferase promoter design - (Aug/22/2006 )

Hi all

I am new in the field of promoter studies, so that's why im dropping a few questions here.

I already have a luciferase vector (PGL3.1), but i dont know what part of the promoter i have to clone into the MCS. I am particularly interested in one specific bindingsite, so that should be included for sure. But where to start en where to end?

Is there anyone who can give me some useful advise (or a paper)?


Thanks!


Madscientist

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I haven't actually made my own reporter but I've used a few different ones we received from other groups. The ones I've used seem to "start" at least a kilobase upstream of the transcriptional site (-1000) and go up to the transcriptional start site or a little further downstream (+1 to +50). Alot of people make nested sets of reporters getting progressively shorter to look at the activity of particular sites which can then be mutated by site-directed mutagenesis to confirm their activity. It's probably a good idea to look in the literature. Has anyone cloned your gene promoter before or the promoter of a similar/related gene? If so that's probably a good place to start.

All the best,
Ceri

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I think the pGL3 already has a SV40 promoter to express luciferase. U need not clone another one in.

Verify with the vector map.

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It depends on the PGL3 he is using: some are for enhancer studies, therefore you have already a promoter and you clone your enhancer of interest; some are for promoter studies and you have to clone your own promoter.

I would suggest to search for the paper from Stiwie and Putzer, in which they cloned the TAp73 promoter including the E2F-1 binding site

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I am thinking of using the pgl3.1 basic, without the SV40 promoter and use my own promoter. I wanna activate the promoter by overexpreesing a gene that is likely to express the gene behind the promoter.

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This is exactly what I do. I would advice you again to have a look at Stiwie's paper about TAp73.

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