ArticleAntibodies (Basel, Switzerland)2019
A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells.
Article in Antibodies (Basel, Switzerland), 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 21 citations in OpenAlex.
- Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems.Frontiers in molecular biosciences · 2022Article
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- A light way for nuclear cell biologists.Journal of biochemistry · 2021Review
- Protein manipulation using single copies of short peptide tags in cultured cells and inDevelopment (Cambridge, England) · 2021Article
- Nanobodies Right in the Middle: Intrabodies as Toolbox to Visualize and Modulate Antigens in the Living Cell.Biomolecules · 2020Review
- An Inside Job: Applications of Intracellular Single Domain Antibodies.Biomolecules · 2020Review
- Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology.BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy · 2020Review
- Special Issue: Nanobody.Antibodies (Basel, Switzerland) · 2020Article
- A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo.Nature communications · 2019Article
- Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components.Frontiers in synaptic neuroscience · 2019Review
Corrections and comments
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Authors and funding
6 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.