I have found a few mutations in the upstream region of my gene of interest, and I would like to test in vitro whether they have a role in the regulation of the expression of this gene. I have done heaps of cloning, in vitro work, luciferase assays, etc for miRNA work but I am not so experienced with upstream regulatory regions. I would appreciate some help with the following:
-what plasmid would be the best to use?
- with the cloning, do I clone the whole region, or a small region containing each mutation?
- I assume I would need to make a plasmid containing the same region but without the mutations to use as controls?
- Do I need to co-transfect the cells with anything (for example, a transcription factor predicted to bind to this region)?
This kind of experiments are well suited with pGL series of plasmids specifically pGL3 basic. It has firefly luciferase without promoter. In place for promoter (upstream of luciferase gene) you'll clone the mutant sequence. To choose the region, copy paste the entire upstream sequence (about 2kb) upstream of start codon and check it in promoterscan site (http://www-bimas.cit.nih.gov/molbio/proscan/). Look for regions that has clusters of transcription factor binding sites and TATA box and clone that region into pGL3 basic. From this cloned vector, you can create wild type sequences by site directed mutagenesis to make mutant bases into wild type). All these plasmids will need to be transfected (transient) with pRL-TK (which has renila luciferase TK promoter...but you can choose CMV promoter containing pRL vectors too). Then you'll do dual luciferase assay to see which mutations regulate the expression of firefly luciferase (in other words, your target gene). The pRL vector will be used to normalize transfection efficiency. Good Luck.
Thanks a lot Goodwin, I will have a look at the methods you suggested.
BTW Addgene has loads of pGL3 derivatives that you might find useful as controls or backbones.
https://www.addgene.org/search/advanced/?q=pgl3
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