Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human beta -globin locus in erythropoietic cells.
Reactivation of fetal hemoglobin genes has been proposed as a potential therapeutic procedure in patients with beta-thalassemia, sickle cell disease, or other beta-hemoglobinopathies. In vitro model systems based on small plasmid globin gene constructs have previously been used in human and mouse erythroleukemic cell lines to study the molecular mechanisms regulating the expression of the fetal human globin genes and their reactivation by a variety of pharmacologic agents. These studies have led to great insights in globin gene regulation and the identification of a number of potential inducers of fetal hemoglobin. In this study we describe the development of enhanced green fluorescence protein (EGFP) reporter systems based on bacterial artificial chromosomes (EBACs) to monitor the activity of the epsilon-, (G)gamma-, (A)gamma-, delta-, and beta-globin genes in the beta-globin locus. Additionally, we demonstrate that transfection of erythroleukemia cells with our EBACs is greatly enhanced by expression of EBNA1, which also facilitates episomal maintenance of our constructs in human cells. Our studies in human cells have shown physiologically relevant differences in the expression of each of the globin genes and also demonstrate that hemin is a potent inducer of EGFP expression from EGFP-modified epsilon-, (G)gamma-, and (A)gamma-globin constructs. In contrast, the EGFP-modified delta- and beta-globin constructs consistently produced much lower levels of EGFP expression on hemin induction, mirroring the in vivo ontogeny. The EGFP-modified beta-globin eukaryotic BAC (EBAC) vector system can thus be used in erythroleukemia cells to evaluate induction of the epsilon- and gamma-globin genes from the intact human beta-globin locus.
Vadolas
Jim
J
Cell and Gene Therapy Research Group, The Murdoch Children's Research Institute, Royal Children's Hospital, Melbourne, Australia.
Wardan
Hady
H
Orford
Michael
M
Voullaire
Lucille
L
Zaibak
Faten
F
Williamson
Robert
R
Ioannou
Panayiotis A
PA
D002454:Cell Differentiation; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000166:cytology / Q000378:metabolism*; D005319:Fetal Hemoglobin / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006427:Hemin / Q000494:pharmacology; D006801:Humans; D020014:K562 Cells; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D012680:Sensitivity and Specificity; D014162:Transfection
D019309:Epstein-Barr Virus Nuclear Antigens; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D006427:Hemin; D005914:Globins; D005319:Fetal Hemoglobin; C417029:EBV-encoded nuclear antigen 1