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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2001-12-0365.
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Blood, 1 December 2002, Vol. 100, No. 12, pp. 4209-4216
RED CELLS
Development of sensitive fluorescent assays for embryonic and
fetal hemoglobin inducers using the human  -globin locus in
erythropoietic cells
Jim Vadolas,
Hady Wardan,
Michael Orford,
Lucille Voullaire,
Faten Zaibak,
Robert Williamson, and
Panayiotis A. Ioannou
From the Cell and Gene Therapy Research Group, The
Murdoch Children's Research Institute, Royal Children's Hospital,
Melbourne, Australia; and the Cyprus Institute of
Neurology and Genetics, Nicosia, Cyprus.
Reactivation of fetal hemoglobin genes has been proposed as a
potential therapeutic procedure in patients with  -thalassemia, sickle cell disease, or other -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
 -, G -, A-,  -, and -globin genes
in the -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 -, G-, and
A-globin constructs. In contrast, the EGFP-modified -
and -globin constructs consistently produced much lower levels of
EGFP expression on hemin induction, mirroring the in vivo ontogeny. The
EGFP-modified -globin eukaryotic BAC (EBAC) vector system can
thus be used in erythroleukemia cells to evaluate induction of the
- and -globin genes from the intact human -globin locus.
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