Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro
Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro
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DOI：
10.1006/mthe.2000.0075
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发表时间：
2000-06-01
影响因子：
12.4
通讯作者：
Kay, MA
中科院分区：
文献类型：
--
作者：
Miao, CH;Ohashi, K;Kay, MA
我们系统地比较了人因子IX基因表达的各种质粒含有不同的顺式调控序列后，转染到不同的肝细胞系，或在体内，注射到小鼠的肝脏。尽管培养细胞的基因表达存在1.5至2.0倍的变异，但在体内研究中观察到65倍的变异。我们发现，含有载脂蛋白E基因座控制区（HCR）、人α 1-抗胰蛋白酶（hAAT）启动子、hFIX小基因（hFIX mg）序列（包括第一内含子的一部分）的质粒，（内含子A）、3 '-非翻译区（3'-UTR）和牛生长激素多聚腺苷酸化信号（bpA）产生最高血清水平的人因子IX，注射后1天达到18 μ g/ml（正常= 5 μ g/ml）。尽管大多数质粒DNA导致瞬时基因表达，但包含内含子、来自1.7-kb 3 '-UTR或0.3-kb bpA的多聚腺苷酸化信号和HCR导致hFIX基因表达的持续和治疗水平，范围为0.5 - 2 μ g/ml这些数据强调了顺式序列对于增强体内肝基因表达的重要性，并再次强调了组织培养和体内研究中基因表达缺乏相关性。
We systematically compared human factor IX gene expression from a variety of plasmids containing different cis-regulatory sequences after transfection into different hepatocyte cell lines, or in vivo, after their injection into the livers of mice. Although there was a 1.5- to 2.0-fold variation in gene expression from cultured cells, a 65-fold variation was observed in the In vivo studies. We found that a plasmid containing the apolipoprotein E locus control region (HCR), human alpha1-antitrypsin (hAAT) promoter, hFIX minigene (hFIXmg) sequence including a portion of the first intron (intron A), 3'-untranslated region (3'-UTR), and a bovine growth hormone polyadenylation signal (bpA) produced the highest serum level of human factor IX, reaching 18 mug/ml (normal = 5 mug/ml) 1 day after injection. Although most of the plasmid DNAs resulted in transient gene expression, inclusion of an intron, a polyadenylation signal from either the 1.7-kb 3'-UTR or the 0.3-kb bpA, and the HCR resulted in persistent and therapeutic levels of hFIX gene expression, ranging from 0.5 to 2 mug/ml (10 to 40% of normal) for 225 days (length of experiment), These data underscore the importance of cis sequences for enhancing in vivo hepatic gene expression and reemphasize the lack of correlation of gene expression in tissue culture and in vivo studies.