Kinetic analysis and modeling of firefly luciferase as a quantitative reporter gene in live mammalian cells.
Firefly luciferase has proven to be a highly sensitive and quantitative reporter gene for studying gene delivery and regulation, and its recent use in live cells and organisms promises to further expand its utility. However, the intracellular behavior and properties of the enzyme are not well characterized. Specifically, information on the intracellular kinetics and stability of luciferase activity is necessary for real-time luminescence counts from live cells to be quantitatively meaningful. Here, we report a dynamic analysis of luciferase activity in the context of living mammalian cells. We have determined the relative light units measured in living cells to be proportional to that found in cell lysate. We have also calculated the K(m) of luciferase in living cells to be approximately 1 mM, a value much higher than the 10 microM found for pure enzyme in vitro. In addition, a 2-hour half-life of luciferase activity in live cells was measured in real time. Finally, we have modeled luciferase activity in live cells for the purposes of understanding and translating the luciferase signal into a more effective metric of gene expression and cell behavior.
Ignowski
Jolene M
JM
Department of Chemical Engineering and The Helen Wills Neuroscience Institute, MC 1462, University of California-Berkeley, Berkeley, CA 94720-1462, USA.
Schaffer
David V
DV
D000818:Animals; D002460:Cell Line; D048888:Fireflies / Q000201:enzymology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D042541:Intracellular Space / Q000648:ultrastructure; D007700:Kinetics; D008156:Luciferases / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008962:Models, Theoretical
D008156:Luciferases