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.

Expression and Misexpression of the miR-183 Family in the Developing Hearing Organ of the Chicken

“…pGFP was first modified to create a Gateway destination vector (pT2K-CAG-EGFP-attR) by inserting the attR cassette (Invitrogen) into the EcoRV site located after the EGFP coding sequence. An approximately 800 base pair fragment containing genomic sequences from the mouse miR-183 family locus, flanked by splice donor and acceptor sites, was obtained from pME-MCS-sd-miR183F-sa [ 30 ]. The miR-183 intron was inserted into pT2K-CAG-EGFP-attR through a Gateway LR recombination reaction, creating pT2K-CAG-EGFP-183F (abbreviated pGFP-183F).…”

“…The miR-183 intron was inserted into pT2K-CAG-EGFP-attR through a Gateway LR recombination reaction, creating pT2K-CAG-EGFP-183F (abbreviated pGFP-183F). Plasmid pT2K-CAG-EGFP-9 (abbreviated pGFP-9) was created by inserting miR-9 coding sequence from pME-MCS-sd-9-sa [ 30 ] into pT2K-CAG-EGFP-attR and was used as a control in luciferase assays.…”

“…Plasmids (miRNA reporters and pGFP, pGFP-183F, or pGFP-9) were transfected using Lipofectamine 2000. Details regarding construction of miRNA reporters and the luciferase assays are published [ 30 ]. After 24 hours the cells were lysed and luciferase activity was assessed using the dual luciferase assay kit (Promega) in concert with the Luminoskan Ascent luminometer (Thermo Fisher).…”

Tol2-Mediated Delivery of miRNAs to the Chicken Otocyst Using Plasmid Electroporation

“…We maintained about 60 nucleotides of endogenous sequence on the 5′ and 3′ ends of pre-miR-9-1. We also had success with constructs containing a larger number of fl anking bases [ 25 ].…”

“…We inserted the amplicon containing the artifi cial intron into a Gateway ® shuttle vector to create pME-MCS-sda as described previously [ 25 ]. We placed three restriction enzyme sites ( Xba I, Bam HI, and Xho I) in the artifi cial intron to facilitate entry of any miRNA of interest.…”