A versatile microsatellite instability reporter system in human cells.
A versatile microsatellite instability reporter system in human cells.
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DOI：
10.1093/nar/gkt615
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发表时间：
2013-09
影响因子：
14.9
通讯作者：
Tijsterman M
中科院分区：
文献类型：
--
作者：
Koole W;Schäfer HS;Agami R;van Haaften G;Tijsterman M
在这里，我们报告的调查微卫星不稳定性（MSI）在人类细胞与新开发的报告系统的基础上荧光。我们组成了一个载体，其中可以在荧光蛋白mCherry的功能拷贝和EGFP的框外拷贝之间引入不同长度和核苷酸组成的微卫星;体内移码将导致EGFP表达，其可以通过荧光激活细胞分选（FACS）进行定量。通过靶向重组工程，将单拷贝报告基因引入HEK 293和MCF-7细胞中。我们发现主要是-1和+1碱基对移码，其水平通过错配修复保持一致。我们发现，道的长度和组成极大地影响MSI。相比之下，片段形成G-四链体结构的潜力、其链方向或其转录状态不影响MSI。我们进一步验证了报告系统用于筛选化合物的微卫星致突变性和鉴定MSI修饰剂的功能：使用逆转录病毒miRNA表达文库，我们鉴定了靶向MSH 2的miR-21作为过表达时诱导MSI的miRNA。我们的数据还为将荧光报告基因与下一代测序技术相结合以识别特定途径中的遗传因素的策略提供了原理证明。
Here, we report the investigation of microsatellite instability (MSI) in human cells with a newly developed reporter system based on fluorescence. We composed a vector into which microsatellites of different lengths and nucleotide composition can be introduced between a functional copy of the fluorescent protein mCherry and an out-of-frame copy of EGFP; in vivo frameshifting will lead to EGFP expression, which can be quantified by fluorescence activated cell sorting (FACS). Via targeted recombineering, single copy reporters were introduced in HEK293 and MCF-7 cells. We found predominantly −1 and +1 base pair frameshifts, the levels of which are kept in tune by mismatch repair. We show that tract length and composition greatly influences MSI. In contrast, a tracts’ potential to form a G-quadruplex structure, its strand orientation or its transcriptional status is not affecting MSI. We further validated the functionality of the reporter system for screening microsatellite mutagenicity of compounds and for identifying modifiers of MSI: using a retroviral miRNA expression library, we identified miR-21, which targets MSH2, as a miRNA that induces MSI when overexpressed. Our data also provide proof of principle for the strategy of combining fluorescent reporters with next-generation sequencing technology to identify genetic factors in specific pathways.
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DOI：
10.1038/nrg3296
发表时间：
2012-11
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
通讯作者：
--
影响因子：
82.9
作者：
通讯作者：
--
影响因子：
9.8
作者：
Brinkmann, B;Klintschar, M;Rolf, B
通讯作者：
Rolf, B
影响因子：
10.7
作者：
Beck, NR;Double, MC;Cockburn, A
通讯作者：
Cockburn, A
影响因子：
64.5
作者：
BOSHART, M;WEBER, F;SCHAFFNER, W
通讯作者：
SCHAFFNER, W