Chromosomal translocations induced at specified loci in human stem cells
Chromosomal translocations induced at specified loci in human stem cells
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DOI：
10.1073/pnas.0902076106
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发表时间：
2009-06-30
影响因子：
11.1
通讯作者：
Jasin, Maria
中科院分区：
文献类型：
--
作者：
Brunet, Erika;Simsek, Deniz;Jasin, Maria
干细胞和前体细胞的精确基因操作为分析、预防和治疗人类恶性肿瘤提供了非凡的潜力。染色体易位是几种肿瘤类型的特征，它们被认为出现在干细胞或前体细胞中。虽然已经有方法研究小鼠细胞易位形成的因素，但在人类细胞中，特别是在相关细胞类型中，方法一直缺乏。锌指核酸酶(ZFN)技术允许DNA双链断裂(DSB)被引入特定的染色体座位。我们利用这项技术在两个内源性基因座上同时产生DSB，在19号染色体上产生PPP1R12C/P84基因，在X染色体上产生IL2R伽马基因，从而在人类细胞中诱导染色体易位。用高通量96孔巢式定量聚合酶链式反应，利用变性曲线和DNA测序，在多种人类细胞类型中检测t(19；X)易位断点连接，包括胚胎干细胞(HES)和HES细胞来源的间充质前体细胞。虽然易位很容易被发现，但与通过基因打靶或非同源末端连接修复单个DSB相比，易位的频率较低，这两种方法都不会导致严重的染色体重排。虽然以前的研究依赖于繁琐的细胞遗传修饰和培养中的广泛生长，但本报告中描述的方法很容易适用于原代人类细胞，包括多能和多能细胞，以揭示靶向易位和其他基因组重排的潜在机制和表型后果。
The precise genetic manipulation of stem and precursor cells offers extraordinary potential for the analysis, prevention, and treatment of human malignancies. Chromosomal translocations are hallmarks of several tumor types where they are thought to have arisen in stem or precursor cells. Although approaches exist to study factors involved in translocation formation in mouse cells, approaches in human cells have been lacking, especially in relevant cell types. The technology of zinc finger nucleases (ZFNs) allows DNA double-strand breaks (DSBs) to be introduced into specified chromosomal loci. We harnessed this technology to induce chromosomal translocations in human cells by generating concurrent DSBs at 2 endogenous loci, the PPP1R12C/p84 gene on chromosome 19 and the IL2R gamma gene on the X chromosome. Translocation breakpoint junctions for t(19;X) were detected with nested quantitative PCR in a high throughput 96-well format using denaturation curves and DNA sequencing in a variety of human cell types, including embryonic stem (hES) cells and hES cell-derived mesenchymal precursor cells. Although readily detected, translocations were less frequent than repair of a single DSB by gene targeting or nonhomologous end-joining, neither of which leads to gross chromosomal rearrangements. While previous studies have relied on laborious genetic modification of cells and extensive growth in culture, the approach described in this report is readily applicable to primary human cells, including mutipotent and pluripotent cells, to uncover both the underlying mechanisms and phenotypic consequences of targeted translocations and other genomic rearrangements.