Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells.
Targeting of the human coagulation factor IX gene at rDNA locus of human embryonic stem cells.
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人类凝血因子 IX 基因靶向人类胚胎干细胞 rDNA 位点
DOI：
10.1371/journal.pone.0037071
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Liang D
中科院分区：
文献类型：
--
作者：
Liu X;Wu Y;Li Z;Yang J;Xue J;Hu Y;Feng M;Niu W;Yang Q;Lei M;Xia J;Wu L;Liang D
基因修饰是实现人类胚胎干细胞（hESCs）在人类遗传研究和再生医学中的全部潜力的先决条件。不幸的是，随机整合方法一直是使用的主要技术不断产生问题，而主要的替代方法，基因靶向，在操纵小鼠胚胎干细胞（mESCs）方面有效，但在hESCs方面效果不佳。人类核糖体DNA （rDNA）重复序列聚集在顶中心染色体的短臂上。它们由每个单倍体大约400个45S前rna （rRNA）基因组成。在本研究中，我们通过同源重组将人凝血因子IX生理基因定位到hESCs的rDNA位点。相对基因靶向效率（>50%）和同源重组频率（>10−5）比以往报道的靶向位点高10倍以上。同时，目标克隆既保留了正常的核型，又保留了胚胎干细胞的主要特征。该基因在靶向hESCs中稳定且异位表达。结论/意义这是首次将人类生理基因定位于hESC基因组的特定位点。我们的研究结果表明，rDNA位点可能是hESCs中转基因的理想避风港。
Background Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs) in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs) but poorly in hESCs. Methodology/Principal Findings Human ribosomal DNA (rDNA) repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA) gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50%) and homologous recombination frequency (>10−5) were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs. Conclusion/Significance This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs.
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