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Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences

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...

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Autores principales: Stoller, Michelle L., Chang, Henry C., Fekete, Donna M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794778/
https://www.ncbi.nlm.nih.gov/pubmed/24013374
http://dx.doi.org/10.3390/ijms140918239
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author Stoller, Michelle L.
Chang, Henry C.
Fekete, Donna M.
author_facet Stoller, Michelle L.
Chang, Henry C.
Fekete, Donna M.
author_sort Stoller, Michelle L.
collection PubMed
description 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.
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spelling pubmed-37947782013-10-21 Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences Stoller, Michelle L. Chang, Henry C. Fekete, Donna M. Int J Mol Sci Article 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. MDPI 2013-09-05 /pmc/articles/PMC3794778/ /pubmed/24013374 http://dx.doi.org/10.3390/ijms140918239 Text en © 2013 by the authors; licensee MDPI, Basel, Switzerland http://creativecommons.org/licenses/by/3.0 This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).
spellingShingle Article
Stoller, Michelle L.
Chang, Henry C.
Fekete, Donna M.
Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences
title Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences
title_full Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences
title_fullStr Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences
title_full_unstemmed Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences
title_short Bicistronic Gene Transfer Tools for Delivery of miRNAs and Protein Coding Sequences
title_sort bicistronic gene transfer tools for delivery of mirnas and protein coding sequences
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794778/
https://www.ncbi.nlm.nih.gov/pubmed/24013374
http://dx.doi.org/10.3390/ijms140918239
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