Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.
Bottom Line: Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells.Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B.We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.Show MeSH
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Mentions: Having validated this set of AURKB-GFP cells generated from the same parental HeLa line, we systematically analyzed the behavior of the expressed fusion proteins. First, we examined total expression level of AURKB-GFP by Western blotting of whole-cell extracts with anti-AURKB or anti-GFP antibodies (Figure 2A and Supplemental Figure S2A). All genome-edited clones exhibited AURKB-GFP levels similar to the endogenous protein with relatively little variation and independent of the genome-targeting method used to generate them or their zygosity (∼0.6- to 1.7-fold wild type). By contrast, expression of AURKB-GFP from randomly integrated BACs resulted in clearly visible overexpression in the tested clones (∼2.1- to 3.6-fold wild type), suggesting that the presence of the native promoter is not sufficient to ensure physiological expression levels. As expected for a constitutive heterologous promoter (cytomegalovirus; Boshart et al., 1985), different cDNA clones showed the strongest overexpression and the most variable expression between cell lines (∼0.4– to 5.7-fold). Flow cytometry of the intensity distribution of AURKB-GFP from 20,000 cells for each tagging method showed greatest cell-to-cell homogeneity for the two genome-editing methods (CV = 23 [ZFN], 23 [TALEN]), followed by BACs and cDNAs, which exhibited more variability (Figure 2B; CV = 30 and 112, respectively). Similarly, whereas in genome-edited and BAC systems, nearly all cells expressed the fusion protein (ZFN, 99%; BAC, 92–99%), cDNA clones had a variable number of expressing cells (Figure 2B; 28–89%).
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.