title: Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation.
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 (G)γ- or (A)γ-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.
authors.affiliation: Cell and Gene Therapy Group, Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia.
authors.forename: Kasey S K
authors.initials: KS
authors.lastname: Chan
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.forename: Hady
authors.initials: H
authors.lastname: Wardan
authors.forename: Bradley
authors.initials: B
authors.lastname: McColl
authors.forename: Stuart
authors.initials: S
authors.lastname: Orkin
authors.forename: Jim
authors.initials: J
authors.lastname: Vadolas
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D005319:Fetal Hemoglobin / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D012097:Repressor Proteins; D019076:Transgenes; D055544:beta-Globins / Q000235:genetics*; D055545:gamma-Globins / Q000235:genetics*
chemical_list: C486563:BCL11A protein, human; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D009687:Nuclear Proteins; D012097:Repressor Proteins; D055544:beta-Globins; C485184:enhanced green fluorescent protein; D055545:gamma-Globins; D049452:Green Fluorescent Proteins; D005319:Fetal Hemoglobin