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Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences
Department of Biological Sciences, Purdue University, 915 W State St, West Lafayette, IN 47907-1392, USA
Purdue University Center for Cancer Research, Purdue University, 201 S University Dr, West Lafayette, IN 47907-2064, USA
* Author to whom correspondence should be addressed.
Received: 22 July 2013; in revised form: 8 August 2013 / Accepted: 13 August 2013 / Published: 5 September 2013
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.
Keywords: miRNAs; gene therapy; miR-183 family
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MDPI and ACS Style
Stoller, M.L.; Chang, H.C.; Fekete, D.M. Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences. Int. J. Mol. Sci. 2013, 14, 18239-18255.
Stoller ML, Chang HC, Fekete DM. Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences. International Journal of Molecular Sciences. 2013; 14(9):18239-18255.
Stoller, Michelle L.; Chang, Henry C.; Fekete, Donna M. 2013. "Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences." Int. J. Mol. Sci. 14, no. 9: 18239-18255.