Two-Color Imaging of Nonrepetitive Endogenous Loci in Human Cells.,ACS Synthetic Biology

当前位置： X-MOL 学术 › ACS Synth. Biol. › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Two-Color Imaging of Nonrepetitive Endogenous Loci in Human Cells.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-08-21 , DOI: 10.1021/acssynbio.0c00295
Ipek Tasan _{1,

2} , Christina Jean Su ₃ , Behnam Enghiad _{2,

4} , Meng Zhang _{2,

4} , Shekhar Mishra _{2,

4} , Huimin Zhao _{1,

2,

4}

Affiliation

Tools for live cell imaging of multiple nonrepetitive genomic loci in mammalian cells are necessary to study chromatin dynamics. Here, we report a new system based on two chromosomally integrated orthogonal irregular repeat arrays and particularly a new general strategy to construct irregular repeat arrays. Briefly, we utilized a “bridge oligonucleotide-mediated ligation” protocol to assemble 8-mer repeats de novo which were then combined into a final 96-mer repeat array using Golden Gate cloning. This strategy was used for assembling a new mutant TetO irregular repeat array, which worked orthogonally to the wild type TetO repeat. Single copy integration of the new repeat array did not cause replication deficiencies at the tagged locus. Moreover, the mutant TetO irregular repeat could also be visualized by CRISPR imaging. Our new irregular repeat assembly method demonstrates a generally applicable strategy that can be used for assembling additional orthogonal repeat arrays for imaging genomic loci and irregular repeats to visualize RNA or proteins via signal amplification.

中文翻译：

人类细胞中非重复性内源基因座的双色成像。

哺乳动物细胞中多个非重复基因组位点的活细胞成像工具是研究染色质动力学所必需的。在这里，我们报告了一个基于两个染色体整合的正交不规则重复阵列的新系统，特别是构建不规则重复阵列的新通用策略。简而言之，我们利用“桥接寡核苷酸介导的连接”方案从头组装 8 聚体重复序列然后使用 Golden Gate 克隆将其组合成最终的 96 聚体重复阵列。该策略用于组装新的突变体 TetO 不规则重复阵列，该阵列与野生型 TetO 重复阵列正交工作。新重复阵列的单拷贝整合不会导致标记位点的复制缺陷。此外，突变的 TetO 不规则重复也可以通过 CRISPR 成像可视化。我们新的不规则重复组装方法展示了一种普遍适用的策略，可用于组装额外的正交重复阵列，用于成像基因组位点和不规则重复，以通过信号放大可视化 RNA 或蛋白质。

更新日期：2020-09-20

点击分享查看原文

点击收藏

阅读更多本刊最新论文本刊介绍/投稿指南11