Hello everyone,
In actually I am new in this concept. I want to create plasmid system for dual luciferase assay(promega) for spesisfic gene and mutant version of it. For example P53 and several mutation on it. I want to see the effect of mutation on transcriptional activitiy.
For this what kind of insert can I use, how can I design it. I have some gaps in my mind. Anyone can suggest to me same paper or rewiev about it ?
Thank you.
Dear Mustafa, are you referring to the Dual Luciferase systems using SRF-RE, SRE, NFAT-RE or CRE promoters?
The ones i know of, they have these promoters followed by one luciferase gene (for ex, firefly), and one CMV or other constitutive promoter followed by another luciferase gene. The CMV part is for normalizing cell number.
To study a gene of interest, for example p53, you should transfect the cells with both the Dual Luciferase plasmid and your gene of interest (wild type or mutated; also maybe knockout the endogeneous p53 if needed) and then stimulate the cells in such a way that your gene (p53) will be used to elicit a transcriptional response. I do not work with p53 but as far as i know it is a transcription factor. You should find out which DNA sequences p53 can bind to and activate gene expression.
If the transcriptional response element is one of the 4 above, you can use the appropriate plasmid immediately. If it is some other element, you could use cloning to modify the plasmid and insert your desired promoter instead of the original one (SRE or the others), however, you will have to do some optimizations.
Dear Sara Mingu,
Thank you for your answer. It helped me better understand some points.Do you know which promoter has whether advantage to study mutational effect of interested gene? I also read about this promoters and usages, if u have another source, could u share with me please.
My best regards,
Thank you,
Dear Mustafa, in this paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5478243/ in section 2.7 they talk about some potential p53 binding sites which they have cloned to a similar promega plasmid, maybe some can be appropriate for you.
I suggest you check p53 binding sites which are present in your cell or tissue site if possible. Also you should choose a sequence which is activated (rather than repressed) by p53.
This article https://www.ncbi.nlm.nih.gov/pmc/articles/PMC187891/ or referenced ones may hve more information about these sequences and they have also made a similar luciferase reporter.
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