Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells
Robert Mahen, Birgit Koch, Malte Wachsmuth, Antonio Z Politi, Alexis Perez-Gonzalez, Julia Mergenthaler, Yin Cai, Jan Ellenberg
MOLECULAR BIOLOGY OF THE CELL | AMER SOC CELL BIOLOGY | Published : 2014
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 ima..View full abstract
Awarded by European Commission within the MitoSys and SystemsMicroscopy consortia
Awarded by EMBO fellowship
Awarded by Wellcome Trust Henry Wellcome Fellowship
We thank Tony Hyman and Ina Poser for donating the mouse BAC AURKB-GFP and thank all members of the Ellenberg laboratory for support. We thank Kota Miura for help in setting up the automated Fiji analysis and Bianca Nijmeijer for help with Southern blotting. This study was supported by funding from the European Commission within the MitoSys and SystemsMicroscopy consortia (FP7/2007-2013-241548 and FP7/2007-2013-258068). R. M. was supported by an EMBO fellowship (ALTF 416-2012, GA-2010-267146) and a Wellcome Trust Henry Wellcome Fellowship (100090/Z/12/Z).