Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.
Human beta-globin gene expression in transgenic mice is enhanced by a distant DNase I hypersensitive site.
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DOI：
10.1073/pnas.86.18.7082
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发表时间：
1989-09
影响因子：
11.1
通讯作者：
Peter T. Curtin;Depei Liu;Wen Liu;JudyC. Chang;Yuet Wai Kan
中科院分区：
文献类型：
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作者：
Peter T. Curtin;Depei Liu;Wen Liu;JudyC. Chang;Yuet Wai Kan
多项证据表明，位于人类 β-珠蛋白基因上游的红细胞特异性 DNase I 超敏位点 (HS) 对于调节 β-珠蛋白基因表达非常重要。我们使用聚合酶链式反应技术来扩增和克隆跨越这些 HS 之一的 882 个碱基对 DNA 片段，指定为 HSII，它位于 β-珠蛋白基因上游 54 KB 处。克隆的 HSII 片段以基因组 (HSII-beta) 或反基因组 (HSII-beta) 方向与人 β-珠蛋白基因连接。将这两种构建体和单独的β-珠蛋白基因（β）注射到受精小鼠卵中，并分析第16天转基因胎儿的肝脏和大脑中的表达。 7 个 β 转基因胎儿中有 5 个表达人 β 珠蛋白 mRNA，但每个基因拷贝的表达水平较低，为小鼠 α 珠蛋白 mRNA 的 0.93% 至 22.4%（平均 9.9%）。相比之下，12 个 HSII-β 转基因胎儿中有 11 个每个基因拷贝的 β-珠蛋白 mRNA 表达水平为小鼠 α-珠蛋白 mRNA 的 31.3% 至 336.6%（平均 139.5%）。仅产生了三个含有完整 HSII-β 构建体拷贝的胎儿。三个中的两个以每个基因拷贝的 179.2% 和 387.1% 的水平表达人类 β-珠蛋白 mRNA。人β-珠蛋白 mRNA 的表达在所有三种类型的转基因胎儿中均具有组织特异性。这些研究表明，含有单个红细胞特异性 HS 的小 DNA 片段可以刺激转基因小鼠中高水平的人 β-珠蛋白基因表达。
Several lines of evidence suggest that erythroid-specific DNase I hypersensitive sites (HS) located far upstream of the human beta-globin gene are important in regulating beta-globin gene expression. We used the polymerase chain reaction technique to amplify and clone an 882-base-pair DNA fragment spanning one of these HS, designated HSII, which is located 54 kilobases upstream of the beta-globin gene. The cloned HSII fragment was linked to a human beta-globin gene in either the genomic (HSII-beta) or antigenomic (HSII-beta) orientation. These two constructs and a beta-globin gene alone (beta) were injected into fertilized mouse eggs, and expression was analyzed in liver and brain from day-16 transgenic fetuses. Five of 7 beta-transgenic fetuses expressed human beta-globin mRNA, but the level of expression per gene copy was low, ranging from 0.93 to 22.4% of mouse alpha-globin mRNA (average 9.9%). In contrast, 11 of 12 HSII-beta transgenic fetuses expressed beta-globin mRNA at levels per gene copy ranging from 31.3 to 336.6% of mouse alpha-globin mRNA (average 139.5%). Only three fetuses containing intact copies of the HSII-beta construct were produced. Two of three expressed human beta-globin mRNA at levels per gene copy of 179.2 and 387.1%. Expression of human beta-globin mRNA was tissue-specific in all three types of transgenic fetuses. These studies demonstrate that a small DNA fragment containing a single erythroid-specific HS can stimulate high-level human beta-globin gene expression in transgenic mice.