( G) FACS analysis of H2B-BFP or BFP-LMNA expression in HeLa cells. ( F) Bar graph showing the size of dCas9-GFP 14X spots representing H2B or LMNA loci, with and without transcription, respectively. ( E) Statistics of signal-to-noise ratio to demonstrate DNA labeling efficiency at H2B and LMNA loci with or without transcription respectively, n ≥ 97 genomic loci. ( D) Statistics of signal-to-noise ratio (SNR) to report the labeling of nascent transcripts at H2B and LMNA loci, n ≥ 90 genomic loci. ( C) Line scan of the relative fluorescence of the signal indicated by the dotted lines in (C). ( B) Representative images to show simultaneous visualization of DNA, RNA and protein of H2B (top and middle) or LMNA (bottom) genes in HeLa cells. A hybrid of MS2 loops and CRISPR-Tag was first generated and then inserted in the intron of blue fluorescent protein (BFP). Tricolor visualization of DNA, RNA, and protein in living cells using TriTag. Published by Oxford University Press on behalf of Nucleic Acids Research. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation.
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