Cargando…
A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells
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 i...
Autores principales: | , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640688/ https://www.ncbi.nlm.nih.gov/pubmed/31544816 http://dx.doi.org/10.3390/antib8010010 |
_version_ | 1783436623291088896 |
---|---|
author | Keller, Bettina-Maria Maier, Julia Weldle, Melissa Segan, Soeren Traenkle, Bjoern Rothbauer, Ulrich |
author_facet | Keller, Bettina-Maria Maier, Julia Weldle, Melissa Segan, Soeren Traenkle, Bjoern Rothbauer, Ulrich |
author_sort | Keller, Bettina-Maria |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6640688 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-66406882019-09-05 A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells Keller, Bettina-Maria Maier, Julia Weldle, Melissa Segan, Soeren Traenkle, Bjoern Rothbauer, Ulrich Antibodies (Basel) Article 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. MDPI 2019-01-10 /pmc/articles/PMC6640688/ /pubmed/31544816 http://dx.doi.org/10.3390/antib8010010 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Keller, Bettina-Maria Maier, Julia Weldle, Melissa Segan, Soeren Traenkle, Bjoern Rothbauer, Ulrich A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells |
title | A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells |
title_full | A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells |
title_fullStr | A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells |
title_full_unstemmed | A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells |
title_short | A Strategy to Optimize the Generation of Stable Chromobody Cell Lines for Visualization and Quantification of Endogenous Proteins in Living Cells |
title_sort | strategy to optimize the generation of stable chromobody cell lines for visualization and quantification of endogenous proteins in living cells |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640688/ https://www.ncbi.nlm.nih.gov/pubmed/31544816 http://dx.doi.org/10.3390/antib8010010 |
work_keys_str_mv | AT kellerbettinamaria astrategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT maierjulia astrategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT weldlemelissa astrategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT segansoeren astrategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT traenklebjoern astrategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT rothbauerulrich astrategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT kellerbettinamaria strategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT maierjulia strategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT weldlemelissa strategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT segansoeren strategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT traenklebjoern strategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells AT rothbauerulrich strategytooptimizethegenerationofstablechromobodycelllinesforvisualizationandquantificationofendogenousproteinsinlivingcells |