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Bicistronic gene transfer tools for delivery of miRNAs and protein coding sequences.

Stoller ML, Chang HC, Fekete DM - Int J Mol Sci (2013)

Bottom Line: Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway.The genes downstream are successfully translated into protein and identifiable through immunofluorescence.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 915 W State St, West Lafayette, IN 47907-1392, USA. dfekete@purdue.edu.

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.

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Plasmid containing miR-183 family and the GFP gene produces functional miRNAs and GFP protein in HEK293T cells. (A) Vector design of p183F-GFP; (B) Visualization of GFP in cells transfected with p183F-GFP. Scale bar = 100 microns; (C) The miR-183 family is expressed from p183F-GFP in mammalian and avian cells. Cells transfected with p183F-GFP showed expression of mature miR-183, -96, and -182. Control (untransfected cells) and pGFP transfected cells exhibit no detectable miRNA. U6 levels serve as the loading control; (D–F) Luciferase activity is decreased by expression of miRNAs from p183F-GFP expressing vector; (D) Cells co-transfected with p183F-GFP and the psiCHECK-2 reporter containing sites complementary to miR-183 show a significant decrease in luciferase activity compared to cells co-transfected with the pGFP and reporter; (E) and (F) show results of experiments similar to (D) except the reporter contained different complementary binding sites: 96 for (E) and 182 for (F). Each bar represents mean (±standard error) for each group. Each experiment was replicated at least three times. *p < 0.05, **p < 0.005, ***p < 0.0001.
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f5-ijms-14-18239: Plasmid containing miR-183 family and the GFP gene produces functional miRNAs and GFP protein in HEK293T cells. (A) Vector design of p183F-GFP; (B) Visualization of GFP in cells transfected with p183F-GFP. Scale bar = 100 microns; (C) The miR-183 family is expressed from p183F-GFP in mammalian and avian cells. Cells transfected with p183F-GFP showed expression of mature miR-183, -96, and -182. Control (untransfected cells) and pGFP transfected cells exhibit no detectable miRNA. U6 levels serve as the loading control; (D–F) Luciferase activity is decreased by expression of miRNAs from p183F-GFP expressing vector; (D) Cells co-transfected with p183F-GFP and the psiCHECK-2 reporter containing sites complementary to miR-183 show a significant decrease in luciferase activity compared to cells co-transfected with the pGFP and reporter; (E) and (F) show results of experiments similar to (D) except the reporter contained different complementary binding sites: 96 for (E) and 182 for (F). Each bar represents mean (±standard error) for each group. Each experiment was replicated at least three times. *p < 0.05, **p < 0.005, ***p < 0.0001.

Mentions: While our primary interest involved the dual expression of the miR-183 family and Atoh1, it was also desirable to express the miR-183 family alone to assess how much of an impact this family can have on HC development by themselves. To accomplish this, the Atoh1-HA coding region was replaced with GFP, which would allow the identification of cells expressing transfected miRNA constructs. Furthermore, as the design of p183F-GFP is the same as the p183F-Atoh1-HA (Figure 5A), phenotypic analysis using vectors with or without Atoh1-HA is less likely to be confounded by changes in the processing of the RNA transcripts that may affect transcript levels.


Bicistronic gene transfer tools for delivery of miRNAs and protein coding sequences.

Stoller ML, Chang HC, Fekete DM - Int J Mol Sci (2013)

Plasmid containing miR-183 family and the GFP gene produces functional miRNAs and GFP protein in HEK293T cells. (A) Vector design of p183F-GFP; (B) Visualization of GFP in cells transfected with p183F-GFP. Scale bar = 100 microns; (C) The miR-183 family is expressed from p183F-GFP in mammalian and avian cells. Cells transfected with p183F-GFP showed expression of mature miR-183, -96, and -182. Control (untransfected cells) and pGFP transfected cells exhibit no detectable miRNA. U6 levels serve as the loading control; (D–F) Luciferase activity is decreased by expression of miRNAs from p183F-GFP expressing vector; (D) Cells co-transfected with p183F-GFP and the psiCHECK-2 reporter containing sites complementary to miR-183 show a significant decrease in luciferase activity compared to cells co-transfected with the pGFP and reporter; (E) and (F) show results of experiments similar to (D) except the reporter contained different complementary binding sites: 96 for (E) and 182 for (F). Each bar represents mean (±standard error) for each group. Each experiment was replicated at least three times. *p < 0.05, **p < 0.005, ***p < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3794778&req=5

f5-ijms-14-18239: Plasmid containing miR-183 family and the GFP gene produces functional miRNAs and GFP protein in HEK293T cells. (A) Vector design of p183F-GFP; (B) Visualization of GFP in cells transfected with p183F-GFP. Scale bar = 100 microns; (C) The miR-183 family is expressed from p183F-GFP in mammalian and avian cells. Cells transfected with p183F-GFP showed expression of mature miR-183, -96, and -182. Control (untransfected cells) and pGFP transfected cells exhibit no detectable miRNA. U6 levels serve as the loading control; (D–F) Luciferase activity is decreased by expression of miRNAs from p183F-GFP expressing vector; (D) Cells co-transfected with p183F-GFP and the psiCHECK-2 reporter containing sites complementary to miR-183 show a significant decrease in luciferase activity compared to cells co-transfected with the pGFP and reporter; (E) and (F) show results of experiments similar to (D) except the reporter contained different complementary binding sites: 96 for (E) and 182 for (F). Each bar represents mean (±standard error) for each group. Each experiment was replicated at least three times. *p < 0.05, **p < 0.005, ***p < 0.0001.
Mentions: While our primary interest involved the dual expression of the miR-183 family and Atoh1, it was also desirable to express the miR-183 family alone to assess how much of an impact this family can have on HC development by themselves. To accomplish this, the Atoh1-HA coding region was replaced with GFP, which would allow the identification of cells expressing transfected miRNA constructs. Furthermore, as the design of p183F-GFP is the same as the p183F-Atoh1-HA (Figure 5A), phenotypic analysis using vectors with or without Atoh1-HA is less likely to be confounded by changes in the processing of the RNA transcripts that may affect transcript levels.

Bottom Line: Many of these miRNAs are found within introns and processed through the RNA polymerase II pathway.The genes downstream are successfully translated into protein and identifiable through immunofluorescence.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).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Purdue University, 915 W State St, West Lafayette, IN 47907-1392, USA. dfekete@purdue.edu.

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.

Show MeSH