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During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.  相似文献   

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Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to β-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase β-globin promoter-Renilla luciferase β-globin yeast artificial chromosome (γ-luc β-luc β-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no β-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.  相似文献   

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We established a mouse Ltk- cell line that contains within its genome a herpes simplex virus thymidine kinase gene (tk) that had been disrupted by the insertion of the recognition sequence for yeast endonuclease I-SceI. The artificially introduced 18 bp I-SceI recognition sequence was likely a unique sequence in the genome of the mouse cell line. To assess whether an induced double-strand break (DSB) in the genomic tk gene would be repaired preferentially by gene targeting or non-homologous recombination, we electroporated the mouse cell line with endonuclease I-SceI alone, one of two different gene targeting constructs alone, or with I-SceI in conjunction with each of the two targeting constructs. Each targeting construct was, in principle, capable of correcting the defective genomic tk sequence via homologous recombination. tk+ colonies were recovered following electroporation of cells with I-SceI in the presence or absence of a targeting construct. Through the detection of small deletions at the I-SceI recognition sequence in the mouse genome, we present evidence that a specific DSB can be introduced into the genome of a living mammalian cell by yeast endonuclease I-SceI. We further report that a DSB in the genome of a mouse Ltk- cell is repaired preferentially by non-homologous end-joining rather than by targeted homologous recombination with an exogenous donor sequence. The potential utility of this system is discussed.  相似文献   

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The origin of DNA replication in the human β-globin gene contains an initiation region (IR) and two flanking auxiliary elements. Two replicator modules are located within the upstream auxiliary sequence and the IR core, but the functional sequences in the downstream auxiliary element are unknown. Here, we use a combination of benzoylated-naphthoylated DEAE (BND) cellulose purification and nascent strand abundance assays to show that replication initiation occurs at the β-globin 3′ enhancer on human chromosome 11 in the Hu11 hybrid murine erythroleukemia (MEL) cell line. To examine replicator function, 3′ enhancer fragments were inserted into an ectopic site in MEL cells via an optimized FRT/EGFP-FLP integration system. These experiments demonstrate that the 1.6 kb downstream auxiliary element is a third replicator module called bGRep-E in erythroid cells. The minimal 260 bp 3′ enhancer is required but not sufficient to initiate efficient replication, suggesting cooperation with adjacent sequences. The minimal 3′ enhancer also cooperates with elements in an expressing HS3β/γ-globin construct to initiate replication. These data indicate that the β-globin replicator has multiple initiation sites in three closely spaced replicator modules. We conclude that a mammalian enhancer can cooperate with adjacent sequences to create an efficient replicator module.  相似文献   

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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.  相似文献   

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The DNaseI hypersensitive sites (HSs) of the human β-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human β-globin locus yeast artificial chromosome (β-YAC) lines were produced in which the ε-globin gene was replaced with a second marked β-globin gene (βm), coupled to an intact LCR, a 5′HS3 complete deletion (5′ΔHS3) or a 5′HS3 core deletion (5′ΔHS3c). The 5′ΔHS3c mice expressed βm-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type β-globin was co-expressed in adult mice. Although the 5′HS3 core was not required for βm-globin expression, previous work showed that the 5′HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5′HS complete deletion mice, except βm-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.  相似文献   

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