title: Bicistronic gene transfer tools for delivery of miRNAs and protein coding sequences.
abstract: MicroRNAs (miRNAs) are a category of small RNAs that modulate levels of proteins via post-transcriptional inhibition. Currently, a standard strategy to overexpress miRNAs is as mature miRNA duplexes, although this method is cumbersome if multiple miRNAs need to be delivered. Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway. We have designed a vector to exploit this naturally-occurring intronic pathway to deliver the three members of the sensory-specific miR-183 family from an artificial intron. In one version of the vector, the downstream exon encodes the reporter (GFP) while another version encodes a fusion protein created between the transcription factor Atoh1 and the hemaglutinin epitope, to distinguish it from endogenous Atoh1. In vitro analysis shows that the miRNAs contained within the artificial intron are processed and bind to their targets with specificity. The genes downstream are successfully translated into protein and identifiable through immunofluorescence. More importantly, Atoh1 is proven functional through in vitro assays. These results suggest that this cassette allows expression of miRNAs and proteins simultaneously, which provides the opportunity for joint delivery of specific translational repressors (miRNA) and possibly transcriptional activators (transcription factors). This ability is attractive for future gene therapy use.
authors.affiliation: Department of Biological Sciences, Purdue University, 915 W State St, West Lafayette, IN 47907-1392, USA. dfekete@purdue.edu.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Stoller
authors.forename: Henry C
authors.initials: HC
authors.lastname: Chang
authors.forename: Donna M
authors.initials: DM
authors.lastname: Fekete
mesh_terms: D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D002460:Cell Line; D006801:Humans; D007438:Introns / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics*; D010957:Plasmids / Q000235:genetics
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D035683:MicroRNAs; C500942:ATOH1 protein, human; C533449:MIRN183 microRNA, human