cahill-harnessingintronic-2022.pdf (6.47 MB)
Harnessing Intronic microRNA Structures to Improve Tolerance and Expression of shRNAs in Animal Cells
journal contribution
posted on 2022-02-10, 00:00 authored by A Challagulla, M L Tizard, T J Doran, David CahillDavid Cahill, K A JenkinsExogenous RNA polymerase III (pol III) promoters are commonly used to express short hairpin RNA (shRNA). Previous studies have indicated that expression of shRNAs using standard pol III promoters can cause toxicity in vivo due to saturation of the native miRNA pathway. A potential way of mitigating shRNA-associated toxicity is by utilising native miRNA processing enzymes to attain tolerable shRNA expression levels. Here, we examined parallel processing of exogenous shRNAs by harnessing the natural miRNA processing enzymes and positioning a shRNA adjacent to microRNA107 (miR107), located in the intron 5 of the Pantothenate Kinase 1 (PANK1) gene. We developed a vector encoding the PANK1 intron containing miR107 and examined the expression of a single shRNA or multiple shRNAs. Using qRT-PCR analysis and luciferase assay-based knockdown assay, we confirmed that miR30-structured shRNAs have resulted in the highest expression and subsequent transcript knockdown. Next, we injected Hamburger and Hamilton stage 14–15 chicken embryos with a vector encoding multiple shRNAs and confirmed that the parallel processing was not toxic. Taken together, this data provides a novel strategy to harness the native miRNA processing pathways for shRNA expression. This enables new opportunities for RNAi based applications in animal species such as chickens.
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Journal
Methods and ProtocolsVolume
5Issue
1Article number
18Pagination
1 - 13Publisher
MDPI / MDPI AG (Multidisciplinary Digital Publishing Institute)Location
Basel, SwitzerlandPublisher DOI
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2409-9279Language
engPublication classification
C1 Refereed article in a scholarly journalUsage metrics
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