Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing.
Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing.
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DOI：
10.1038/srep35949
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发表时间：
2016-10-26
影响因子：
4.6
通讯作者：
Cissé II
中科院分区：
文献类型：
--
作者：
Cho WK;Jayanth N;Mullen S;Tan TH;Jung YJ;Cissé II
哺乳动物RNA聚合酶II（Pol II）的活细胞成像以前依赖于随机插入外源性突变Pol II，并使用毒素α-鹅膏蕈碱降解内源性Pol II。因此，目前尚不清楚标记的Pol II在外源启动子下的过表达是否可能在体内报告的Pol II动力学中发挥作用。在这里，我们使用CRISPR/Cas9基因编辑系统标记小鼠胚胎成纤维细胞（MEF）中的内源性Pol II。在活细胞中使用基于单分子的超分辨率成像，我们捕获了内源性Pol II簇。与以前的研究一致，我们观察到Pol II簇在活细胞中的寿命很短（簇寿命~8 s）。此外，动态响应血清刺激，药物介导的转录抑制都与以前的观察结果在外源性Pol II MEF细胞系。我们的研究结果表明，以前的外源性标记的Pol II忠实地概括了活细胞中的内源性聚合酶聚类动力学，我们的方法原则上可用于直接标记转录因子进行活细胞成像。
Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging.
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