Abstract
Single-domain antibodies have emerged as highly versatile nanoprobes for advanced cellular imaging. For real-time visualization of endogenous antigens, fluorescently labelled nanobodies (chromobodies, CBs) are introduced as DNA-encoded expression constructs in living cells. Commonly, CB expression is driven from strong, constitutively active promoters. However, high expression levels are sometimes accompanied by misfolding and aggregation of those intracellular nanoprobes. Moreover, stable cell lines derived from random genomic insertion of CB-encoding transgenes bear the risk of disturbed cellular processes and inhomogeneous CB signal intensities due to gene positioning effects and epigenetic silencing. In this study we propose a strategy to generate optimized CB expressing cell lines. We demonstrate that expression as ubiquitin fusion increases the fraction of intracellularly functional CBs and identified the elongation factor 1α (EF1-α) promoter as highly suited for constitutive CB expression upon long-term cell line cultivation. Finally, we applied a CRISPR/Cas9-based gene editing approach for targeted insertion of CB expression constructs into the adeno-associated virus integration site 1 (AAVS1) safe harbour locus of human cells. Our results indicate that this combinatorial approach facilitates the generation of fully functional and stable CB cell lines for quantitative live-cell imaging of endogenous antigens.
Highlights
Cell culture models provide substantial information on various cellular responses ranging from exposure to small chemical compounds to genetically mediated target depletion [1]
This approach is limited to the visualization of ectopically expressed fluorescent protein (FP) fusions, which may considerably differ from their endogenous counterparts in terms of expression level, activity, localization and protein half-life [4,5,6,7,8]
Besides the generation of proteins with a desired N-terminus, the ubiquitin fusion technique has been reported to increase solubility and functionality of ectopically expressed proteins [30]. According to these findings we noticed that ubiquitin fusions of CBs are less prone to aggregation irrespective of the adjacent amino acid. Whether these potential benefits can be transferred to CBs such as the lamin-specific CB (LMN-CB), which has previously been shown to form aggregates upon high expression levels in HeLa cells [23,31]
Summary
Cell culture models provide substantial information on various cellular responses ranging from exposure to small chemical compounds to genetically mediated target depletion [1]. For imaging-based analysis, expression of fluorescent fusion proteins (FP fusions) is the most commonly used approach to study localization and dynamic changes of proteins in living cells. Available FP-based live-cell assays enable the investigation of processes such as cell proliferation, apoptosis or DNA damage. This approach is limited to the visualization of ectopically expressed FP fusions, which may considerably differ from their endogenous counterparts in terms of expression level, activity, localization and protein half-life [4,5,6,7,8]. Evolved gene editing methods such as the Antibodies 2019, 8, 10; doi:10.3390/antib8010010 www.mdpi.com/journal/antibodies
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