Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin.
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 gamma and beta 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 gamma globin gene inducers are recognized by their ability to increase gamma-firefly luciferase (gamma(F)) gene activity significantly more than beta-renilla luciferase (beta(R)) gene activity, identified by an increased ratio of gamma-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 gamma-globin gene expression. It can therefore be used for the screening of chemical agents that may have gamma-globin gene inducibility.
Skarpidi
E
E
Division of Medical Genetics, University of Washington, Seattle, WA 98195, USA.
Vassilopoulos
G
G
Li
Q
Q
Stamatoyannopoulos
G
G
D000818:Animals; D001120:Arginine / Q000494:pharmacology; D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line; D001517:Coleoptera; D003545:Cysteine / Q000494:pharmacology; D004275:DNA, Ribosomal; D005319:Fetal Hemoglobin / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005914:Globins / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D011392:Proline / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D037401:Scyphozoa; D014162:Transfection
D002087:Butyrates; D004275:DNA, Ribosomal; D011422:Propionates; D011993:Recombinant Fusion Proteins; D005914:Globins; D005319:Fetal Hemoglobin; D001120:Arginine; D011392:Proline; D008156:Luciferases; D003545:Cysteine