Abstract
Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human γ and β globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific γ globin gene inducers are recognized by their ability to increase γ-firefly luciferase (γF) gene activity significantly more than β-renilla luciferase (βR) gene activity, identified by an increased ratio of γ-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase γ-globin gene expression. It can therefore be used for the screening of chemical agents that may have γ-globin gene inducibility.
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