MUC4 is not expressed in cell lines used for live cell imaging.
MUC4 is not expressed in cell lines used for live cell imaging.
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DOI：
10.12688/wellcomeopenres.17229.2
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发表时间：
2021
影响因子：
--
通讯作者：
Bickmore WA
中科院分区：
文献类型：
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作者：
Athmane N;Williamson I;Boyle S;Biddie SC;Bickmore WA
背景：可视化活细胞中特定哺乳动物基因位点的能力对于理解与转录相关的动态过程非常重要。然而，据报道，一些用于靶向哺乳动物基因进行活细胞成像的工具，如dCas9，本身会阻碍与转录相关的过程。MUC4基因是活细胞成像研究的热门靶标，因为该基因的一些外显子内的序列具有重复性。方法：我们开始比较dCas9和基于tale的成像工具对MUC4表达的影响，包括在先前报道表达MUC4的人类细胞系中。结果：我们无法在这些细胞系中检测到MUC4 mRNA。此外，对与转录相关的组蛋白修饰的公开数据和转录本身的数据的分析表明，在dCas9靶向抑制MUC4和MUC1表达的细胞系中，或在dCas13用于报道活细胞中MUC4 RNA检测的细胞系中，MUC4和任何粘蛋白基因家族都没有显著表达。结论：可视化人类活细胞中特定基因位点和基因转录物的方法非常具有挑战性。我们的数据表明，应该谨慎选择最合适的细胞系进行这些分析，并仔细比较正交分析基因表达的方法。
Background: The ability to visualise specific mammalian gene loci in living cells is important for understanding the dynamic processes linked to transcription. However, some of the tools used to target mammalian genes for live cell imaging, such as dCas9, have been reported to themselves impede processes linked to transcription. The MUC4 gene is a popular target for live cell imaging studies due to the repetitive nature of sequences within some exons of this gene. Methods: We set out to compare the impact of dCas9 and TALE-based imaging tools on MUC4 expression, including in human cell lines previously reported as expressing MUC4. Results: We were unable to detect MUC4 mRNA in these cell lines. Moreover, analysis of publicly available data for histone modifications associated with transcription, and data for transcription itself, indicate that neither MUC4, nor any of the mucin gene family are significantly expressed in the cell lines where dCas9 targeting has been reported to repress MUC4 and MUC1 expression, or in the cell lines where dCas13 has been used to report MUC4 RNA detection in live cells. Conclusions: Methods for visualising specific gene loci and gene transcripts in live human cells are very challenging. Our data suggest that care should be given to the choice of the most appropriate cell lines for these analyses and that orthogonal methods of assaying gene expression be carefully compared.