Reporter-promoter construct and transfection. I want to design such a construct for checking the activity of AP1. I would also like to know if calcium phosphate method of transfection is good enough for transfection mammalian cells because I am finding lipofectamine to be a very costly option.
These days you can get promoter-reporters commercially, please see link below (on the MAPK/JNK AP1 Response Element (AP1 RE) pNL[NlucP/AP1-RE/Hygro) Vector). Calcium phosphate coprecipitation is a cheap method to achieve acceptable transfection efficiency, I don't have experience with it but I might work better on large adherent fibroblastoid cells compared to other cells.
https://www.promega.com/a/forms/custom-assays/luciferase-pathway-reporters.html
Dear Sierra,
Thanks for your reply. For my understanding, I want to know at which site in a vector do we target (within promoter) to insert the synthesized AP1 -TRE? This designing is also a part of my academic project so I can't buy it commercially?
I have to work on Hep G2 cells? Can I use calcium phosphate. Any one who has an answer is welcome.
Hi. The easiest way to guide you in your design is to download a map of the commercial vector, however you might also just focus on the important elements. If you're using a luciferase reporter, then it's important to verify that you have a basic promoter containing a TATA box and that the LUC coding sequence starts with a good Kozak motif. Having said that, the ideal situation is to clone the AP1 elements upstream of the basic promoter. To have a powerful reporter is better to design your oligos so that when you mix them and ligate them, they concatemerize thus making your reporter with several binding sites. This is usually done by ordering 5'-phosphorylated oligos that create a restrction site when you align them,say that you get a top strand than when you mix it with a bottom strand, both make one restriction site on either end and ready to ligate (with overhangs or receding nucleotides just as if you had cut them) . The tricky part is to find the proper ratio of oligos and vector so that you get 6 or so binding sites sometimes in tandem or they might be inverse. But to avoid all this trouble, and taking into account the lower prices for long oligos these days, you could order a concatemerized long oligo and just stick it in a simple cloning step, and voila. 'Hope this helps.
THANKS FOR YOUR ANSWER OSCAR! It was really helpful. Like you told I ma designing the Transcription response element (TRE) to get it synthesized commercially. However, I got stuck at a point where I am not understanding how do I ligate the TRE upstream of promoter and where exactly withing upstream region.
I would appreciate if you can tell me how do I decide where exactly (exactly where upstream of promoter) I should digest my vector .
thanks
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