Spontaneous switch from Aγ- to β-globin promoter activity in a stable transfected dual reporter vector

Giovanni Migliaccio, Angela Di Baldassarre, Cristina Di Rico, Antonella Di Noia, Betty Nakamoto, Hua Cao, Eva Skarpidi, Anna Rita Migliaccio

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5 Scopus citations


Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ- and β-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1-2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from Aγ- and β-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the β-promoter increased 10-fold more than that driven by the Aγ one, the ratio between Aγ-driven/ (Aγ-driven + β-driven) reporter activity in the cells decreased after 1 month and became similar to the γ/(γ + β) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the Aγ-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the β-promoter. These results suggest that the rapid changes in activity driven by the Aγ- and β-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the Aγ- and β-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.

Original languageEnglish
Pages (from-to)174-180
Number of pages7
JournalBlood Cells, Molecules, and Diseases
Issue number2
StatePublished - 2005
Externally publishedYes


  • Dual Luciferase reporter assay
  • Epigenetic regulation
  • GM979 cell line
  • Hb switch


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