TY - JOUR
T1 - Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation
AU - Chan, Kasey S K
AU - Xu, Jian
AU - Wardan, Hady
AU - McColl, Bradley
AU - Orkin, Stuart
AU - Vadolas, Jim
PY - 2012/4
Y1 - 2012/4
N2 - 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.
AB - 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.
KW - Induction of fetal hemoglobin
KW - RNAi
UR - http://www.scopus.com/inward/record.url?scp=84860893776&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84860893776&partnerID=8YFLogxK
U2 - 10.1096/fj.11-199356
DO - 10.1096/fj.11-199356
M3 - Article
C2 - 22267339
AN - SCOPUS:84860893776
SN - 0892-6638
VL - 26
SP - 1736
EP - 1744
JO - FASEB Journal
JF - FASEB Journal
IS - 4
ER -