Development of a destabilized firefly luciferase enzyme for measurement of gene expression.
Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid increases or decreases in gene expression may not be detected, owing to the accumulation of residual luciferase. Accordingly, the goal of the present study was to develop a luciferase reporter with a reduced functional half-life. This was accomplished by adding a synthetic fragment to the firefly luciferase-coding sequence that encoded the proteolytic "PEST" signal from mouse ornithine decarboxylase. When placed under the control of estrogen response elements and expressed in human breast cancer T-47D cells, the modified luciferase protein (LUCODC-DA) displayed a functional half-life of 0.84 h compared to 3.68 h for the wild-type enzyme. As anticipated, the overall rate of photonic emissions in cells expressing the destabilized luciferase was about sevenfold lower than that of their wild-type counterparts, presumably because of the reduction of steady-state luciferase accumulation. Even so, the photonic activity derived from LUCODC-DA was still sufficient to enable real-time measurements of gene expression in single living cells.
Leclerc
G M
GM
Medical University of South Carolina, Charleston, USA.
Boockfor
F R
FR
Faught
W J
WJ
Frawley
L S
LS
D000818:Animals; D001943:Breast Neoplasms; D003001:Cloning, Molecular; D001517:Coleoptera; D004795:Enzyme Stability; D015870:Gene Expression; D006207:Half-Life; D006801:Humans; D008156:Luciferases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008845:Microinjections; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection; D014407:Tumor Cells, Cultured
D008156:Luciferases