A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.
A CO-FISH assay to assess sister chromatid segregation patterns in mitosis of mouse embryonic stem cells.
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DOI：
10.1007/s10577-013-9358-8
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发表时间：
2013-05
期刊：
影响因子：
--
通讯作者：
Klar AJ
中科院分区：
文献类型：
--
作者：
Sauer S;Burkett SS;Lewandoski M;Klar AJ
姐妹染色单体包含相同的DNA序列，但它们的螺旋利手性和复制历史都是手性的。来自各种模式生物的新证据表明，某些干细胞非随机地分离姐妹染色单体，以保持基因组的完整性或在不对称的细胞分裂中偏向细胞分化。追踪旧的和新合成的DNA链的传统方法通常缺乏对单个染色体的分辨率，并使用卤代胸苷类似物，对快速分裂的细胞具有深刻的细胞毒性作用。在这里，我们提出了一种改进的染色体定位荧光原位杂交(CO-FISH)方法，其中单个染色体及其复制历史的识别是在随后的与染色体特异的DNA探针和PNA端粒探针的杂交步骤中实现的。重要的是，我们解决了BrdU细胞毒性的问题，并表明我们的方法与正常的小鼠ES细胞生物学兼容，不同于最近发表的相关协议。我们的CO-FISH分析结果表明，小鼠7号染色体在ES细胞中的有丝分裂分离是随机的，这与我们实验室以前发表的结果形成了对比，并解决了一个争议。我们直截了当的方案为未来研究来自不同模式生物的体外培养细胞的染色单体分离模式提供了有用的资源。
Sister chromatids contain identical DNA sequence but are chiral with respect to both their helical handedness and their replication history. Emerging evidence from various model organisms suggests that certain stem cells segregate sister chromatids nonrandomly to either maintain genome integrity or to bias cellular differentiation in asymmetric cell divisions. Conventional methods for tracing of old vs. newly synthesized DNA strands generally lack resolution for individual chromosomes and employ halogenated thymidine analogs with profound cytotoxic effects on rapidly dividing cells. Here, we present a modified chromosome orientation fluorescence in situ hybridization (CO-FISH) assay, where identification of individual chromosomes and their replication history is achieved in subsequent hybridization steps with chromosome-specific DNA probes and PNA telomere probes. Importantly, we tackle the issue of BrdU cytotoxicity and show that our method is compatible with normal mouse ES cell biology, unlike a recently published related protocol. Results from our CO-FISH assay show that mitotic segregation of mouse chromosome 7 is random in ES cells, which contrasts previously published results from our laboratory and settles a controversy. Our straightforward protocol represents a useful resource for future studies on chromatid segregation patterns of in vitro-cultured cells from distinct model organisms.
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