title: A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3' untranslated region-mediated gene regulation.
abstract: Regulation of messenger ribonucleic acid (mRNA) subcellular localization, stability and translation is a central aspect of gene expression. Much of this control is mediated via recognition of mRNA 3' untranslated regions (UTRs) by microRNAs (miRNAs) and RNA-binding proteins. The gold standard approach to assess the regulation imparted by a transcript's 3' UTR is to fuse the UTR to a reporter coding sequence and assess the relative expression of this reporter as compared to a control. Yet, transient transfection approaches or the use of highly active viral promoter elements may overwhelm a cell's post-transcriptional regulatory machinery in this context. To circumvent this issue, we have developed and validated a novel, scalable piggyBac-based vector for analysis of 3' UTR-mediated regulation in vitro and in vivo. The vector delivers three independent transcription units to the target genome--a selection cassette, a turboGFP control reporter and an experimental reporter expressed under the control of a 3' UTR of interest. The pBUTR (piggyBac-based 3' UnTranslated Region reporter) vector performs robustly as a siRNA/miRNA sensor, in established in vitro models of post-transcriptional regulation, and in both arrayed and pooled screening approaches. The vector is robustly expressed as a transgene during murine embryogenesis, highlighting its potential usefulness for revealing post-transcriptional regulation in an in vivo setting.
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Arindam
authors.initials: A
authors.lastname: Chaudhury
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Natee
authors.initials: N
authors.lastname: Kongchan
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Jon P
authors.initials: JP
authors.lastname: Gengler
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Vakul
authors.initials: V
authors.lastname: Mohanty
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Audrey E
authors.initials: AE
authors.lastname: Christiansen
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Fachini
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: James F
authors.initials: JF
authors.lastname: Martin
authors.affiliation: Department of Molecular Physiology and Biophysics and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA neilson@bcm.edu.
authors.forename: Joel R
authors.initials: JR
authors.lastname: Neilson
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D002460:Cell Line; D004251:DNA Transposable Elements; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism; D034622:RNA Interference; D020871:RNA Stability; D016601:RNA-Binding Proteins / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D004251:DNA Transposable Elements; D035683:MicroRNAs; D016601:RNA-Binding Proteins
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