Regulated expression of amplified human β globin genes

Abstract

Gene therapy for the β thalassemias and sickle cell anemia will require high levels of expression of human β globin genes. One method to achieve this goal is amplification of globin genes transferred into the stem cells in the bone marrow of these patients. If the amplified genes remain normally regulated, they will then further increase their expression of being induced to differentiate along an erythroid pathway. To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human β globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells. All the G418-resistant clones analyzed acquired and expressed the human β globin gene. By serial passage of the cells in increasing concentrations of methotrexate, the exogenous human β globin genes were stably amplified in all lines, and all increased their globin mRNA expression roughly proportional to their augmented copy number. Most of the clones further increased their β globin expression on addition of an erythroid stimulus (dimethylsulfoxide). These results indicate that globin gene amplification may be useful in increasing globin mRNA expression in further experiments whose goal is gene therapy.