Abstract
The human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative defects of the globins, based on a number of genome variations found in the globin gene clusters. Hereditary persistence of fetal hemoglobin (HPFH) also caused by similar types of genomic alterations can compensate for the loss of adult hemoglobin. Understanding the regulation of the human γ-globin gene expression is a challenge for the treatment of thalassemia. A mouse model that facilitates high-throughput assays would simplify such studies. We have generated a transgenic dual reporter mouse model by tagging the γ- and β-globin genes with GFP and DsRed fluorescent proteins respectively in the endogenous human β-globin locus. Erythroid cell lines derived from this mouse model were tested for their capacity to reactivate the γ-globin gene. Here, we discuss the applications and limitations of this fluorescent reporter model to study the genetic basis of red blood cell disorders and the potential use of such model systems in high-throughput screens for hemoglobinopathies therapeutics.
Highlights
The human b-globin locus spans,70 Kb containing the regulatory sequences of the Locus Control Region (LCR) and the b-like globin genes situated in the same order as they are expressed throughout ontogeny [59- HBE (e) - HBG2 (Gc) - HBG1 (Ac) HBD (d) - HBB (b) -39]
In order to generate a mouse model where the expression of human globin genes can be followed by fluorescence, we introduced the GFP cDNA (EGFP-N1, Clontech) in the first ATG codon of the HBG1 gene (PAC2 vector) and the DsRed cDNA (DsRed2, Clontech) in the first ATG codon of the HBB gene (PAC2 vector), both ending by a stop codon (Figure 1A) without replacing the endogenous transcripts
The same strategy was followed to generate the second transgenic line, which has the GFP fused to the erythroid-specific membrane protein Glycophorin A (GPA) as the c-globin reporter (Figure 1A and B)
Summary
The human b-globin locus spans ,70 Kb containing the regulatory sequences of the Locus Control Region (LCR) and the b-like globin genes situated in the same order as they are expressed throughout ontogeny [59- HBE (e) - HBG2 (Gc) - HBG1 (Ac) HBD (d) - HBB (b) -39]. Mice bearing the entire human b-globin locus have been a useful model to understand developmental expression patterns of the five functional human b-like globin genes [2]. Studies on the human b-globin locus transgenic mice demonstrated expression of c-globin in the embryonic stage as well as in the early fetal liver of the mouse whereas the b-globin gene was expressed in the fetal and adult stages [2]
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