My requirement is GFP reporter to measure the promoter activity qualitatively (instead of lucifrease based quantitative measurement). So, I would like to clone a GFP reporter construct with my target gene promoter. Can some suggest the best vector to choose and any cloning strategies I need to consider?
Hello Loka. You seem to have a vector that is expressing luciferase gene under the control of a promoter of your interest. However, you wish to replace luciferase with gfp. Is my understanding correct?
I think the easiest way will be to insert the promoter in the pEGFP-C1/C2/C3 vectors by replacing the original CMV promoter. If you see the maps, there is always a 5`AseI site and a 3` NheI/ AgeI option. But you should keep in mind that most of the established luciferase reporter vectors (pGL) are modified to avoid/reduce spurious transcription factor binding sites, but I am not sure how much of background signals will be still left after you remove the CMV promoter from pEGFP backbone. So, if you already have a reliable luciferase reporter, replacing the luciferase CDS with GFP will be more safe. Good Luck.
May be this reference is helpful for the first approach
www.addgene.org/38776/
Hi Syed, you are right. I want GFP instead of luciferase as my reporter plasmid.
Hi Manoj, I have a eGFP plasmid with CMV promoter. If i understood correctly, changing the CMV promoter will that solve my need?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning