title: Single-cell analysis reveals heterogeneity in onset of transgene expression from synthetic tetracycline-dependent promoters.
abstract: Synthetic promoters have been designed for mammalian cells to achieve both temporal and quantitative control over transgene expression without interfering with the endogenous cellular network. Routine applications of synthetic expression systems are based on steady-state measurements of gene expression while the mechanism by which these steady-states are realised at the single-cell level has not been investigated. We focused on the elucidation of the kinetics of doxycycline-controlled synthetic modules as a paradigm. Following gene expression in single cells, we observed a gradual increase of transgene expression within the first 48 h after activation, as determined by flow cytometry. Time-lapse microscopy revealed that the onset of transgene expression was highly variable in individual cells. Interestingly, a bidirectional cassette design showed significantly reduced cell-to-cell heterogeneity in expression. Of note, the influence of the cell cycle seems to be negligible, since the onset of expression correlates with cell division in only a minor fraction of the cell population. In contrast, rapid and synchronous transgene expression could be realized using a posttranslational regulation system that relies on ligand-induced stabilization of a tagged protein. Thus, the inherent temporal variability of transcriptionally regulated synthetic transgene expression systems has to be considered for kinetic and correlative experimental applications.
authors.affiliation: Model Systems for Infection and Immunity, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Ulfert
authors.initials: U
authors.lastname: Rand
authors.forename: Jan
authors.initials: J
authors.lastname: Riedel
authors.forename: Upneet
authors.initials: U
authors.lastname: Hillebrand
authors.forename: Danim
authors.initials: D
authors.lastname: Shin
authors.forename: Steffi
authors.initials: S
authors.lastname: Willenberg
authors.forename: Sara
authors.initials: S
authors.lastname: Behme
authors.forename: Frank
authors.initials: F
authors.lastname: Klawonn
authors.forename: Mario
authors.initials: M
authors.lastname: Köster
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Hauser
authors.forename: Dagmar
authors.initials: D
authors.lastname: Wirth
mesh_terms: D000818:Animals; D004318:Doxycycline / Q000494:pharmacology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D018740:Genetic Heterogeneity; D051379:Mice; D008853:Microscopy; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D059010:Single-Cell Analysis / Q000379:methods*; D058615:Synthetic Biology; D059008:Time-Lapse Imaging; D019076:Transgenes / Q000187:drug effects*
chemical_list: D004318:Doxycycline
keywords: Cell cycle; Cell-to-cell expression heterogeneity; Stabilization of ecDHFR fusion proteins; Time-lapse microscopy; Transcriptional versus post-transcriptional regulation