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: To address this issue, we created HeLa cell lines stably expressing C-terminal monomeric enhanced GFP (mEGFP) fusions of the key mitotic kinase Aurora kinase B (AURKB-GFP) using four different transgene methods (Figure 1A). We used genome editing by ZFNs (Bibikova et al., 2003) or TALENs (Miller et al., 2011) to target recombination of EGFP from a donor plasmid containing regions of homology to endogenous loci (Figure 1Ai). In addition, we performed random integration of BACs or cDNA plasmids (Figure 1Aii; Poser et al., 2008). For each transgene method (Figure 1A), we created several monoclonal or pooled HeLa lines stably expressing AURKB-GFP, which we validated with a series of assays (Figure 1B; see Materials and Methods for detailed protocols). Briefly, after introduction of the tag, expressing cells were isolated by fluorescence-activated cell sorting (FACS; Figure 1C) and then screened by fluorescence microscopy for specific localization to mitotic organelles as expected for AURKB (Figure 1D; Terada et al., 1998). We distinguished homozygous from heterozygous clones (AURKB has three alleles in HeLa cells; Landry et al., 2013) for the genome-editing methods (Figure 1B; In vitro) by genomic PCR (Figure 1E and Supplemental Figure S1A), Western blot (Figure 1F and Supplemental Figure S1B), and Southern blot (Figure 1G and Supplemental Figure S1, C and D).
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.