3 - Multiscale Fluorescence Imaging

Summary

Automated fluorescence microscopy–based screening approaches have become a standard tool in systems biology, usually applied in combination with exogenous regulation of gene expression in order to examine and determine gene function. Gain of function can be created by introducing cDNAs encoding the gene of interest that can be either untagged or tagged for the visualization of the recombinant protein and its subcellular localization (e.g., green fluorescent protein [GFP]-tagged; Temple et al., 2009). After the discovery of RNA interference (RNAi) in the late 1990s and the development of mammalian short interfering RNA (siRNA) and short hairpin RNA (shRNA) libraries in the early 2000s, gene knockdown technologies became a mainstream for loss-of-function screens on a large or genome-wide scale (Heintze et al., 2013). To date, genome-wide siRNA libraries are still the main application in genomic high-throughput screening, although key problems of the RNAi technology have become apparent, such as off-target effects, variable levels of knockdown efficiency, resulting in low-level confidence in hits of screening campaigns. In order to overcome these limitations, alternative methods for manipulation of gene expression have been developed and predominantly rely on gene excision.