To use the firefly protein to monitor IKK, Gross altered cell lines to genetically fuse the luciferase protein to IKB (IKappaB), the protein that comes immediately after IKK in the NF-KappaB pathway. When the pathway is enabled, IKK triggers reactions that lead to the degradation of IKB. In cells with genetically altered IKB, the attached luciferase is broken down too, meaning scientists can detect increased IKK activity via decreased light from the cells.
"This is like doing in-vivo pharmacodynamics and pharmacokinetics," says Piwnica-Worms in reference to the sciences that study the effects, distribution and dissipation of drugs. "Traditionally the only ways we could do those kinds of studies were either to test for levels of the drug in the blood or to label the drug with a radioactive tracer.
"In the case of NF-KappaB, there were also methods that monitored IKK activity via changes in the levels of gene activation at the end of the pathway," he notes. "But those took hours to days to deliver results, and our approach works continuously and in real time." In their study, Gross and Piwnica-Worms tested the technique in live mice by transplanting genetically altered tumor cells and by using a technique that inserted the fused IKB/luciferase protein into liver cells only. They are currently working to develop a line of mice with the IKB/luciferase fusion built into its genetic code.
In addition, they showed that the system is not only helpful for learning if a drug is having the desired effect, it can also be used to fine-tune drug dosage for maximum benefit.
"One of the reviewers of our paper suggested that we should use the system to produce a full dos
Source:Washington University School of Medicine