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A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells
- Source :
- Antibodies, Volume 8, Issue 1, Antibodies, Vol 8, Iss 1, p 10 (2019)
- Publication Year :
- 2019
- Publisher :
- Multidisciplinary Digital Publishing Institute, 2019.
-
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 1&alpha<br />(EF1-&alpha<br />) 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.
- Subjects :
- lcsh:Immunologic diseases. Allergy
0301 basic medicine
Transgene
chromobodies
Immunology
Endogeny
compound screening
Article
live-cell imaging
03 medical and health sciences
0302 clinical medicine
Live cell imaging
Drug Discovery
Immunology and Allergy
CRISPR
Gene
Chemistry
nanobodies
Cell biology
Elongation factor
030104 developmental biology
Cell culture
lcsh:RC581-607
cellular models
030217 neurology & neurosurgery
Intracellular
Subjects
Details
- Language :
- English
- ISSN :
- 20734468
- Database :
- OpenAIRE
- Journal :
- Antibodies
- Accession number :
- edsair.doi.dedup.....efe1386411b369b5596b2f644b856812
- Full Text :
- https://doi.org/10.3390/antib8010010