Abstract
Exogenous 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.
Highlights
The discovery of the RNA interference (RNAi) mechanism in eukaryotes has profoundly enabled our ability to understand the function of genes in living cells and organisms [1]
To examine if an exogenous short hairpin RNAs (shRNA) could be expressed by utilising natural miRNA processing pathways, we began by identifying a miRNA that is highly expressed in different tissues of the chicken
We consulted the gallus expression in situ hybridisation analysis (GESHIA) website [25], which has in situ hybridisation data for all known chicken miRNAs
Summary
The discovery of the RNA interference (RNAi) mechanism in eukaryotes has profoundly enabled our ability to understand the function of genes in living cells and organisms [1]. Previous studies have reported that stable expression of shRNAs in vivo can cause severe toxicity issues in mice in multiple tissue types including the heart [11], brain [12,13] and liver [14] These toxicity issues are primarily attributed to the saturation of the natural miRNA pathway with exogenous shRNAs. Because miRNAs play a dynamic role in regulation of gene expression where they act to control cellular and metabolic pathways in a spatiotemporal manner, saturation of the endogenous miRNA pathway can perturb miRNA-dependent regulation of biological processes [15]. Constructs encoding shRNA under the control of promoters and regulatory sequences delivered by either viral [14] or non-viral [18] vectors often result in random integration of multiple copies into the genome These approaches can have downstream consequences to the biology of the animal but to the acceptance, by regulatory bodies and consumers, of the subsequent genetically modified (GM) animal. We demonstrated that the expression levels achieved from this approach are tolerated by the primordial germ cells (PGCs) of developing chicken embryos suggesting that a germlinemodified chicken that carries this shRNA transgene could be successfully generated
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