I want to quantitate the NF-kB activation in AGS cells as a response to bacterial infection. Therefore I am looking for a suitable plasmid with luciferase reporter activity. Is it always necessary to use Renilla luciferase - co-transfection as well? If yes/no, please help me understand why?(I understand that it is easier to do normalization e.g. number of cells or false positive from other unknown factors), Any reasons other than this?
Moreover, is it possible to construct this plasmid with available backbone vector in our lab eg.pcDNA6/3 etc?
Thanks in advance
Hi Sasikala,
I reckon you will find the paper by Plaisance et al. useful (see attachment). It details the methodology for constructing the plasmid. I have successfully used the pGL3-Nf-kb plasmid for transfecting HEK293T and HUVEC cells.
The argument for use of Renilla luciferase is because it is constitutively expressed and consequently is independent of experimental factors. I have always found it useful for normalising my signals, particularly with cells that are not very adherent (e.g HEK-293T) cells. You can use the Promega Dual-luciferase kit with the Stop-and-glo reagent specifically for Renilla.
Article Recombination signal sequence binding protein J?? is constit...
This article by Plaisance ·et al 1997 used restriction enzymes for plasmid reconstruction as we all did in the 90s.
Are you guys still using the same technology?
Hi Sasikala, do you have to use only luciferase reporter construct?
Hi Nurudeen Hassan,
Thanks for the article and information on Renilla luciferase.
Hi Chandra Somasundaram,
What else method is there? How about reporter cell lines (Invivogen) and quantiBlue method? Please comment on pros and cons of using these (NF-kB luciferase and QuantiBlue) methods. It would be helpful if you share about other reliable methods as well. Thanks a lot for the input.
Hi Sasi,
Refer to, Crispr/Cas-9 genome editing technology.
Of course, you can still use pcDNA6/3. I have used this expression plasmid vector successfully in DNA vaccines against viral diseases in commercial animals (Pigs, Cattle) in collaboration with Merial, France (Merial patented it and marketed).
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