Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site.
Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site.
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通过染色体异位位点整合分析三核苷酸重复稳定性。
DOI：
10.1007/978-1-4939-9784-8_8
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Leffak,Michael
中科院分区：
文献类型：
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作者：
Gadgil,RujutaYashodhan;RiderJr,SDean;Lewis,Todd;Barthelemy,Joanna;Leffak,Michael
CNG微卫星束的扩张是几种神经退行性疾病的原因，包括1型肌强直性营养不良症、亨廷顿病和8型脊髓小脑共济失调。在这里，我们发现扩增（CNG）nrepeats不仅易受扩增和收缩的影响，而且在复制应激后容易发生DNA双链断裂。我们描述了构建含有不同长度CNG重复序列的克隆细胞系的一般策略，其中微卫星在HeLa基因组的单个异位重组受体位点上使用酵母FLP重组酶进行整合。我们展示了两种类型的（CTG/CAG）细胞系，其中一种包含双荧光标记基因在（CTG/CAG）重复序列的两侧，另一种不包含。我们发现，长CNG重复序列在这些细胞系暴露于长时间复制应激时容易发生DNA双链断裂（DSBs）。
Expansions of CNG microsatellite tracts are responsible for several neurodegenerative diseases, including myotonic dystrophy type 1, Huntington disease, and spinocerebellar ataxia type 8. Here we show that expanded (CNG)nrepeats are susceptible not only to expansions and contractions, but are prone to DNA double strand breaks following replication stress. We describe a general strategy for the construction of clonal cell lines containing CNG repeats of various lengths, in which the microsatellites are integrated using the yeast FLP recombinase at a single ectopic recombination acceptor site in the HeLa genome. We illustrate two types of (CTG/CAG) cell lines, one of which contains dual fluorescent marker genes flanking the (CTG/CAG) repeat, and one which does not. We show that long CNG repeats are prone to DNA double strand breaks (DSBs) upon exposure of these cell lines to prolonged replication stress.