RED CELLS Development of sensitive fluorescent assays for embryonic and fetal hemoglobin inducers using the human -globin locus in erythropoietic cells

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 EGFPmodified -, 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. (Blood. 2002;100: 4209-4216)