I want to check if promoters of a few genes (from cattle) are active in the cells. I want to clone GFP under the promoters and transfect the cells with the vector. The problem is I don`t know the promoters. How can I identify the promoters? Would it be OK if I clone 200 – 300 bp of 5` untranscribed region assuming the promoters are there?
If you have sequence of 5'-regulatory region of a gene, check the sequence for the presence of "promoter region" cis-elements using programs that are available from NCBI to predict a promoter region. This would be a good start. If it is predicted that the sequence contains "promoter region", next thing would be to test the sequence experimentally using an approach that you mentioned.
I would use at least 2000 bp upstream sequence from the predicted transcriptional start site in the gene to test promoter region experimentally.
I would replace the coding region of your genes of interest by GFP, so you fuse the start codon (ATG) of your genes to the second codon of GFP, and add any polyadenylation signal after the GFP coding region. Don't put any junk DNA between the stop codon and the polyadenylation signal (like polylinkers etc...). 200-300bps are not enough for the promoter, I would at least use 1-2kb. Long distance enhancers are difficult to predict with programs, so a longer piece is best to start with. Once you know which ones work, you can always narrow down the region of importance, but that's another story.
GFP is a poor choice of reporter compared to luciferase or other enzymatic reporters. The dynamic range of expression is much less, especially if you are using microscopy as a read-out of fluorescent intensity. You won't detect weak promoter activity, unless you are willing to assay GFP by Western blotting. Promega's pGL4 series plasmids are the current gold standard for characterizing promoter activity via transfection.
Promoters vary in length by gene, so there is no "one-size fits all" approach that is guaranteed to work. -2kb to the TSS is a good default guess, but many tissue-specific genes have promoters that span 10kb or more. In the absence of genomics data to guide you, you will probably have to perform a truncation series to map the important regions, as Jurgen suggested. For this reason, definitely start with larger fragments first. In addition, you'll want to include a standard promoter such as PGK or UbC (ubiquitin C) as a positive control in your experiments.
I would not recommend including 5' UTR regions; although these might influence mRNA stability, localization, and translational efficiency, you want to try and design your construct to report directly on transcriptional efficiency, and leave these additional regulatory events out of the picture.
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