Visualizing the Genome: Experimental Approaches for Live-Cell Chromatin Imaging

Abstract

Over the years, our vision of the genome has changed from a linear molecule to that of a complex 3D structure that follows specific patterns and possesses a hierarchical organization. Currently, genomics is becoming “four-dimensional”: our attention is increasingly focused on the study of chromatin dynamics over time, in the fourth dimension. Recent methods for visualizing the movements of chromatin loci in living cells by targeting fluorescent proteins can be divided into two groups. The first group requires the insertion of a special sequence into the locus of interest, to which proteins that recognize the sequence are recruited (e.g., FROS and ParB-INT methods). In the methods of the second approach, “programmed” proteins are targeted to the locus of interest (i.e., systems based on CRISPR/Cas, TALE, and zinc finger proteins). In the present review, we discuss these approaches, examine their strengths and weaknesses, and identify the key scientific problems that can be studied using these methods.

Opticool: Cutting-edge transgenic optical tools

“…By far the most popular systems for real-time transcription detection are the MS2 [28,29] and PP7 [30][31][32][33] systems, whereby MS2 coat protein (MCP) and PP7 coat protein (PCP), fused to fluorophores, bind MS2 and PP7 RNA stem loops, respectively. These have been used to generate a broad array of transgenic animals and computational tools to track gene expression timing in live imaging experiments [19,[34][35][36][37][38][39]. In addition to these popular mRNA visualization options, multiple other aptamer-coat protein pairs are currently available for transgenic studies including BglG The two-part aptamer-binding eIF4a [47] provides an intriguing twist on the system, providing a reliable, straight-forward method to label RNA without ubiquitous background fluorescence by fusing the 2 parts to split fluorophores [48,49] (Fig 1E).…”