Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells
| Autor: | Alexis Perez-Gonzalez, Jan Ellenberg, Yin Cai, Birgit Koch, Julia Mergenthaler, Antonio Z. Politi, Robert Mahen, Malte Wachsmuth |
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| Rok vydání: | 2014 |
| Předmět: |
Transgene
Green Fluorescent Proteins Molecular Sequence Data Mitosis Endogeny Biology Transfection Genes Reporter Methods Aurora Kinase B Humans Transgenes Molecular Biology Regulation of gene expression Base Sequence Articles Cell Biology Recombinant Proteins Molecular Imaging Cell biology Gene Expression Regulation Microscopy Fluorescence Molecular imaging Cytokinesis HeLa Cells Plasmids |
| Zdroj: | Molecular Biology of the Cell |
| ISSN: | 1939-4586 1059-1524 |
| DOI: | 10.1091/mbc.e14-06-1091 |
| Popis: | Despite widespread use the extent to which different mammalian transgene methods report on the properties of endogenous proteins has not been systematically compared. This study shows that the choice of fluorescence-tagging method fundamentally influences the ability to image the activity of the mitotic kinase Aurora B. Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells. However, the extent to which different mammalian transgene methods faithfully report on the properties of endogenous proteins has not been studied comparatively. 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. |
| Databáze: | OpenAIRE |
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