ArticlePloS one2016
Bidirectional Promoter Engineering for Single Cell MicroRNA Sensors in Embryonic Stem Cells.
Article in PloS one, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- Advanced computational predictive models of miRNA-mRNA interaction efficiency.Computational and structural biotechnology journal · 2024Article
- 5' Transgenes drive leaky expression of 3' transgenes in Cre-inducible bi-cistronic vectors.Molecular therapy. Methods & clinical development · 2024Article
- Integration of a multi-omics stem cell differentiation dataset using a dynamical model.PLoS genetics · 2023Article
- Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells.Scientific reports · 2017Article
- Dissecting miRNA gene repression on single cell level with an advanced fluorescent reporter system.Scientific reports · 2017Article
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Authors and funding
2 authors.
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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.
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