title: Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.
abstract: 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.
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Mahen
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Koch
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Wachsmuth
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Antonio Z
authors.initials: AZ
authors.lastname: Politi
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Alexis
authors.initials: A
authors.lastname: Perez-Gonzalez
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Mergenthaler
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
authors.forename: Yin
authors.initials: Y
authors.lastname: Cai
authors.affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany ellenberg@embl.de.
authors.forename: Jan
authors.initials: J
authors.lastname: Ellenberg
mesh_terms: D064107:Aurora Kinase B / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008938:Mitosis; D057054:Molecular Imaging; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; C576921:AURKB protein, human; D064107:Aurora Kinase B