A piggyBac-based reporter system for scalable in vitro and in vivo analysis of 3' untranslated region-mediated gene regulation

Nucleic Acids Res. 2014 Jun;42(10):e86. doi: 10.1093/nar/gku258. Epub 2014 Apr 20.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3' Untranslated Regions*
  • Animals
  • Cell Line
  • DNA Transposable Elements*
  • Gene Expression Regulation*
  • Genes, Reporter
  • Genetic Vectors*
  • Humans
  • Mice
  • MicroRNAs / metabolism
  • RNA Interference
  • RNA Stability
  • RNA-Binding Proteins / metabolism


  • 3' Untranslated Regions
  • DNA Transposable Elements
  • MicroRNAs
  • RNA-Binding Proteins