4.4 Article

Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells

Journal

MOLECULAR BIOLOGY OF THE CELL
Volume 25, Issue 22, Pages 3610-3618

Publisher

AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E14-06-1091

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Funding

  1. European Commission within the MitoSys and SystemsMicroscopy consortia [FP7/2007-2013-241548, FP7/2007-2013-258068]
  2. EMBO fellowship [ALTF 416-2012, GA-2010-267146]
  3. Wellcome Trust Henry Wellcome Fellowship [100090/Z/12/Z]
  4. Wellcome Trust [100090/Z/12/Z] Funding Source: Wellcome Trust

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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.

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