TARGETED DELETION OF 5'HS2 OF THE MURINE BETA-GLOBIN LCR REVEALS THAT IT IS NOT ESSENTIAL FOR PROPER REGULATION OF THE BETA-GLOBIN LOCUS
TARGETED DELETION OF 5'HS2 OF THE MURINE BETA-GLOBIN LCR REVEALS THAT IT IS NOT ESSENTIAL FOR PROPER REGULATION OF THE BETA-GLOBIN LOCUS
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DOI：
10.1101/gad.9.18.2203
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发表时间：
1995-09-15
影响因子：
10.5
通讯作者：
GROUDINE, M
中科院分区：
文献类型：
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作者：
FIERING, S;EPNER, E;GROUDINE, M
β-珠蛋白基因座控制区（LCR）是一个复杂的调控元件，对所有顺式连接的β-珠蛋白基因的适当红细胞特异性表达至关重要。在定义LCR的五个超敏位点中，只有5 'HS 2在体外瞬时和稳定测定中以及在转基因小鼠中显示出增加基因表达。因此，5 'HS 2被认为是体内LCR功能的重要元件。我们利用小鼠胚胎干细胞（ES）中的同源重组和衍生小鼠中的表型分析来研究5 'HS 2在小鼠β-珠蛋白基因座中其正常染色体位置的功能。用选择标记基因（Δ HS 2 +neo）替换5 'HS 2导致基因座中所有基因的表达降低2-5倍，并且当在胚胎发生期间首次检测到最5'胚胎珠蛋白基因Ey的表达时，对该基因Ey的影响更显著（10-12倍）。该突变不会改变珠蛋白基因表达的发育时间。当纯合子时，缺失/替换突变在子宫内是致命的，胚胎在卵黄囊和早期胎肝红细胞生成阶段死亡。为了区分由5 'HS 2的缺失引起的表型效应与可归因于选择标记的插入的表型效应，通过在具有同源重组事件的ES细胞中表达FLP位点特异性重组酶来去除选择标记。从这些ES细胞衍生的小鼠（Delta HS 2 Delta neo）显示出突变染色体上所有β样珠蛋白基因的几乎完全表达。这些结果表明，尽管5’HS 2在多种测定中显示出显著的调节活性，但从内源性β-珠蛋白基因座中删除该元件对基因座表达的时间或程度没有显著影响。此外，这一结果强调，当使用同源重组在体内分析复杂的调控元件时，必须去除插入的选择标记，以避免影响突变的表型。
The beta-globin locus control region (LCR) is a complex regulatory element that is essential for the appropriate red cell-specific expression of all cis-linked beta-globin genes. Of the five hypersensitive sites that define the LCR, only 5'HS2 has been shown to augment gene expression in vitro in both transient and stable assays, as well as in transgenic mice. Thus, 5'HS2 has been assumed to be an important element for the function of the LCR in vivo. We have utilized homologous recombination in murine embryonic stem (ES) cells and phenotypic analysis in derived mice to investigate the function of 5'HS2 in its normal chromosomal position in the murine beta-globin locus. Replacement of 5'HS2 with a selectable marker gene (Delta HS2+neo) causes a 2-5-fold reduction in expression of all of the genes in the locus, and a more pronounced effect (10-12-fold) on the most 5' embryonic globin gene, Ey, when expression of this gene is first detectable during embryogenesis. The mutation produces no alterations in the developmental timing of expression of the globin genes. When homozygous, the deletion/replacement mutation is lethal in utero, with the embryos dying during the stage of yolk sac and early fetal liver erythropoiesis. To distinguish phenotypic effects resulting from the deletion of 5'HS2 from those attributable to insertion of the selectable marker, the selectable marker was removed by expressing the FLP site-specific recombinase in ES cells harboring the homologous recombination event. Mice derived from these ES cells (Delta HS2 Delta neo) demonstrated nearly full expression of all the beta-like globin genes on the mutated chromosome. These results indicate that although 5'HS2 demonstrates significant regulatory activities in a variety of assays, deletion of this element from the endogenous beta-globin locus has no significant effect on the timing or extent of expression of the locus. In addition, this result emphasizes that when using homologous recombination to analyze complex regulatory elements in vivo, the inserted selectable marker must be removed to avoid influencing the phenotype of the mutation.