Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation
Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation
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DOI：
10.1096/fj.11-199356
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发表时间：
2012-04-01
期刊：
影响因子：
4.8
通讯作者：
Vadolas, Jim
中科院分区：
文献类型：
--
作者：
Chan, Kasey S. K.;Xu, Jian;Vadolas, Jim
更好地了解人类γ-珠蛋白表达的调控机制，特别是出生后发生的从γ-珠蛋白到β-珠蛋白的转换，将有助于为β-血红蛋白病患者确定新的治疗靶点。为了进一步阐明γ-珠蛋白表达的机制，开发了一种新的基于荧光的细胞报告分析系统。利用同源重组，将两个报告基因DsRed和EGFP分别插入到183-kb的完整人β-珠蛋白基因座中，分别在（G）γ-或（A）γ-珠蛋白启动子和β-珠蛋白启动子的控制下。将修饰的构建体稳定转染到成年鼠红白血病（MEL）细胞和人胚胎或胎儿红白血病（K562）细胞中，允许根据其发育阶段特异性表达快速和同时分析胎儿和成人珠蛋白基因表达。为了证明该系统的实用性，我们在已知胎儿血红蛋白诱导剂存在或不存在的情况下进行了RNA干扰（RNAi）介导的BCL 11 A敲低，并证明了在成人红细胞环境中γ-珠蛋白连接的报告基因的功能性去抑制。我们的研究结果表明，细胞测定系统代表了一种有前途的方法，可以进行遗传和功能基因组研究，以识别和评估与γ-珠蛋白基因抑制相关的关键因素。陈，K. S. K.，徐，J，Wardan，H.，McColl，B.，Orkin，S.，Vadolas，J. Generation of a genomic reporter assay system for analysis of gamma- and beta-globin gene regulation. FASEB J.26，1736-1744（2012）。www.fasebj.org
A greater understanding of the regulatory mechanisms that govern gamma-globin expression in humans, especially the switching from gamma- to beta-globin, which occurs after birth, would help to identify new therapeutic targets for patients with beta-hemoglobinopathy. To further elucidate the mechanisms involved in gamma-globin expression, a novel fluorescent-based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183-kb intact human beta-globin locus under the control of (G)gamma- or (A)gamma-globin promoter and beta-globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage-specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)-mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a gamma-globin-linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with gamma-globin gene suppression.-Chan, K. S. K., Xu, J., Wardan, H., McColl, B., Orkin, S., Vadolas, J. Generation of a genomic reporter assay system for analysis of gamma- and beta-globin gene regulation. FASEB J. 26, 1736-1744 (2012). www.fasebj.org