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Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells.

Cell. 2019-07; 
LiJieru,DongAnkun,SaydaminovaKamola,ChangHill,WangGuanshi,OchiaiHiroshi,YamamotoTakashi,PertsinidisAlexan
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Gene Synthesis WT and Sox2M HMG mutant Sox2 transient expression constructs were assembled in multiple steps. First EF1a promoter and SV40 terminator were amplified from gene-Blocks (Genscript) and inserted into an empty Piggybac vector (Transposagen Bio, PB007), resulting in PB007-EF1a-SV40. Next, Flag-SNAP-Sox2 WT/2M was amplified from gene-Blocks (Genscript) and cloned into PB007- EF1a-SV40, resulting in PB007- EF1a-Flag-SNAP-Sox2 WT/2M-SV40. Get A Quote

摘要

Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge-one in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intra... More

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