title: Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells.
abstract: MicroRNAs have emerged as important markers and regulators of cell identity. Precise measurements of cellular miRNA levels rely traditionally on RNA extraction and thus do not allow to follow miRNA expression dynamics at the level of single cells. Non-invasive miRNA sensors present an ideal solution but they critically depend on the performance of suitable ubiquitous promoters that reliably drive expression both in pluripotent and differentiated cell types. Here we describe the engineering of bidirectional promoters that drive the expression of precise ratiometric fluorescent miRNA sensors in single mouse embryonic stem cells (mESCs) and their differentiated derivatives. These promoters are based on combinations of the widely used CAG, EF1α and PGK promoters as well as the CMV and PGK enhancers. miR-142-3p, which is known to be bimodally expressed in mESCs, served as a model miRNA to gauge the precision of the sensors. The performance of the resulting miRNA sensors was assessed by flow cytometry in single stable transgenic mESCs undergoing self-renewal or differentiation. EF1α promoters arranged back-to-back failed to drive the robustly correlated expression of two transgenes. Back-to-back PGK promoters were shut down during mESC differentiation. However, we found that a back-to-back arrangement of CAG promoters with four CMV enhancers provided both robust expression in mESCs undergoing differentiation and the best signal-to-noise for measurement of miRNA activity in single cells among all the sensors we tested. Such a bidirectional promoter is therefore particularly well suited to study the dynamics of miRNA expression during cell fate transitions at the single cell level.
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Hanna L
authors.initials: HL
authors.lastname: Sladitschek
authors.affiliation: Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Baden-Württemberg, Germany.
authors.forename: Pierre A
authors.identifier: 0000-0003-4939-3418
authors.initials: PA
authors.lastname: Neveu
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D011401:Promoter Regions, Genetic
chemical_list: D035683:MicroRNAs