Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation

Kasey S K Chan, Jian Xu, Hady Wardan, Bradley McColl, Stuart Orkin, Jim Vadolas

Research output: Contribution to journalArticle

17 Scopus citations

Abstract

A greater understanding of the regulatory mechanisms that govern γ-globin expression in humans, especially the switching from γ- to β-globin, which occurs after birth, would help to identify new therapeutic targets for patients with β-hemoglobinopathy. To further elucidate the mechanisms involved in γ-globin expression, a novel fluorescent-based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183-kb intact human β-globin locus under the control of Gγ- or Aγ-globin promoter and β-globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage-specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)-mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a γ-globin-linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ-globin gene suppression.

Original languageEnglish (US)
Pages (from-to)1736-1744
Number of pages9
JournalFASEB Journal
Volume26
Issue number4
DOIs
StatePublished - Apr 1 2012

Keywords

  • Induction of fetal hemoglobin
  • RNAi

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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