Abstract
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.
Highlights
The ability to visualise specific mammalian gene loci in living cells is important for understanding the dynamic processes linked to transcription
90% confluent U2OS were transfected in a 6-well plate with 1μg of plasmid and 3,75μl of lipofectamine and 4 μl of P3000 reagent. dead Cas9 (dCas9)-EGFP or MUC4 targeting transcription-activator like effectors (TALEs)-EGFP were transiently transfected into U2OS cells prior to imaging and RNA extraction
The ability to detect endogenous gene loci in mammalian cells is an important goal and, the ability to study these genes during the act of transcription is key to understanding both the chromatin dynamics associated with transcription and the spatial organisation of these genes relative to the components of the transcriptional machinery
Summary
The ability to visualise specific mammalian gene loci in living cells is important for understanding the dynamic processes linked to transcription. Some of the tools used to target mammalian genes for live cell imaging, such as dCas, 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 dCas 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. 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
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